Huan Nie

Revisiting the molecular evolutionary history of Shigella spp.

The theory that Shigella is derived from multiple independent origins of Escherichia coli (Pupo et al. 2000) has been challenged by recent findings that the virulence plasmids (VPs) and the chromosomes share a similar evolutionary history (Escobar-Paramo et al. 2003), which suggests that an ancestral VP entered an E. coli strain only once, which gave rise to Shigella spp. In an attempt to resolve these conflicting theories, we constructed three phylogenetic trees in this study: a robust chromosomal tree using 23 housekeeping genes from 46 strains of Shigella and enteroinvasive E. coli (EIEC), a chromosomal tree using 4 housekeeping genes from 19 EcoR strains and 46 Shigella/EIEC strains, and a VP tree using 5 genes outside of the VP cell-entry region from 38 Shigella/EIEC strains. Both chromosomal trees group Shigella into three main clusters and five outliers, and strongly suggest that Shigella has multiple origins within E. coli. Most strikingly, the VP tree shows that the VPs from two main Shigella clusters, C1 and C2, are more closely related, which contradicts the chromosomal trees that place C2 and C3 next to each other but C1 at a distance. Additionally, we have identified a complete tra operon of the F-plasmid in the genome sequence of an EIEC strain and found that two other EIEC strains are also likely to possess a complete tra operon. All lines of evidence support an alternative multiorigin theory that transferable diverse ancestral VPs entered diverse origins of E. coli multiple times during a prolonged period of time, resulting in Shigella species with diverse genomes but similar pathogenic properties.

Complete genome sequence of Shigella flexneri 5b and comparison with Shigella flexneri 2a

BackgroundShigella bacteria cause dysentery, which remains a significant threat to public health. Shigella flexneri is the most common species in both developing and developed countries. Five Shigella genomes have been sequenced, revealing dynamic and diverse features. To investigate the intra-species diversity of S. flexneri genomes further, we have sequenced the complete genome of S. flexneri 5b strain 8401 (abbreviated Sf8401) and compared it with S. flexneri 2a (Sf301).ResultsThe Sf8401 chromosome is 4.5-Mb in size, a little smaller than that of Sf301, mainly because the former lacks the SHI-1 pathogenicity island (PAI). Compared with Sf301, there are 6 inversions and one translocation in Sf8401, which are probably mediated by insertion sequences (IS). There are clear differences in the known PAIs between these two genomes. The bacteriophage SfV segment remaining in SHI-O of Sf8401 is clearly larger than the remnants of bacteriophage SfII in Sf301. SHI-1 is absent from Sf8401 but a specific related protein is found next to the pheV locus. SHI-2 is involved in one intra-replichore inversion near the origin of replication, which may change the expression of iut/iuc genes. Moreover, genes related to the glycine-betaine biosynthesis pathway are present only in Sf8401 among the known Shigella genomes.ConclusionOur data show that the two S. flexneri genomes are very similar, which suggests a high level of structural and functional conservation between the two serotypes. The differences reflect different selection pressures during evolution. The ancestor of S. flexneri probably acquired SHI-1 and SHI-2 before SHI-O was integrated and the serotypes diverged. SHI-1 was subsequently deleted from the S. flexneri 5b genome by recombination, but stabilized in the S. flexneri 2a genome. These events may have contributed to the differences in pathogenicity and epidemicity between the two serotypes of S. flexneri.

Human serum N-glycan profiles are age and sex dependent.

BACKGROUND protein glycosylation varies with the physiological and pathological status of the cell. Consequently, analysis of protein-linked glycans has growing importance both in basic glycobiological research and as a potential tool for monitoring the physiological state in humans. DESIGN, SETTING AND PARTICIPANTS a total of 265 healthy northern Chinese men and women were grouped by age and gender. The mean age in males and females was similar. OBJECTIVE the study is aimed to evaluate the effects of the age and gender on the human serum N-glycans profiles in the clinical diagnose of ageing and disease. METHODS the 265 human serum N-glycan profiles were obtained by DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis. Comparison of N-glycan profiles was carried out among the different genders and age groups and the data were analysed with the GeneMapper software. RESULTS age-related changes in the three N-glycan structures (NGA2F, NGA2FB and NA2F) were observed. Interestingly, fucosylation of N-glycans was significantly different (P < 0.0001) between men and women: more core-α-1,6-fucosylated glycans were detected in women, whereas more branching-α-1,3-fucosylated N-glycans were seen in men. CONCLUSIONS the N-glycome profile in serum is gender and age dependent. This should be taken into consideration in the development of serum glycome markers.

Recent advances in sialic acid-focused glycomics.

Recent emergences of glycobiology, glycotechnology and glycomics have been clarifying enormous roles of carbohydrates in biological recognition systems. For example, cell surface carbohydrates existing as glycoconjugates (glycolipids, glycoproteins and proteoglycans) play crucial roles in cell-cell communication, cell proliferation and differentiation, tumor metastasis, inflammatory response or viral infection. In particular, sialic acids (SAs) existing as terminal residues in carbohydrate chains on cell surface are involved in signal recognition and adhesion to ligands, antibodies, enzymes and microbes. In addition, plasma free SAs and sialoglycans have shown great potential for disease biomarker discovery. Therefore, the development of efficient analytical methods for structural and functional studies of SAs and sialylglycans are very important and highly demanded. The problems of SAs and sialylglycans analysis are vanishingly small sample amount, complicated and unstable structures, and complex mixtures. Nevertheless, in the past decade, mass spectrometry in combination with chemical derivatization and modern separation methodologies has become a powerful and versatile technique for structural analysis of SAs and sialylglycans. This review summarizes these recent advances in glycomic studies on SAs and sialylglycans. Specially, derivatization and capturing of SAs and sialylglycans combined with mass spectrometry analysis are highlighted.

Cell surface-specific N-glycan profiling in breast cancer.

Aberrant changes in specific glycans have been shown to be associated with immunosurveillance, tumorigenesis, tumor progression and metastasis. In this study, the N-glycan profiling of membrane proteins from human breast cancer cell lines and tissues was detected using modified DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). The N-glycan profiles of membrane proteins were analyzed from 7 breast cancer cell lines and MCF 10A, as well as from 100 pairs of breast cancer and corresponding adjacent tissues. The results showed that, compared with the matched adjacent normal tissue samples, two biantennary N-glycans (NA2 and NA2FB) were significantly decreased (p <0.0001) in the breast cancer tissue samples, while the triantennary glycan (NA3FB) and a high-mannose glycan (M8) were dramatically increased (p = 0.001 and p <0.0001, respectively). Moreover, the alterations in these specific N-glycans occurred through the oncogenesis and progression of breast cancer. These results suggested that the modified method based on DSA-FACE is a high-throughput detection technology that is suited for analyzing cell surface N-glycans. These cell surface-specific N-glycans may be helpful in recognizing the mechanisms of tumor cell immunologic escape and could be potential targets for new breast cancer drugs.

Recent Advances in the Synthesis and Biomedical Applications of Chain-End Functionalized Glycopolymers

Glycopolymers as multivalent clusters of carbohydrate derivatives have been proven effective tools in the study of carbohydrate-based biological processes and have shown great potential in biomedical applications. It has been found that the shape and size of glycopolymers, as well as the density and relative positioning of their glycan appendages, are very important regarding their effectiveness in bio-interactions. Recently, a variety of chain-end functionalized polymers have been explored for the preparation of structurally well-defined glycopolymers that have potential protein modification and microarray fabrication applications. This review summarizes recent advances in the synthesis and biomedical applications of chain-end functionalized glycopolymers.

Specific N-glycans of Hepatocellular Carcinoma Cell Surface and the Abnormal Increase of Core-α-1, 6-fucosylated Triantennary Glycan via N-acetylglucosaminyltransferases-IVa Regulation

Glycosylation alterations of cell surface proteins are often observed during the progression of malignancies. The specific cell surface N-glycans were profiled in hepatocellular carcinoma (HCC) with clinical tissues (88 tumor and adjacent normal tissues) and the corresponding serum samples of HCC patients. The level of core-α-1,6-fucosylated triantennary glycan (NA3Fb) increased both on the cell surface and in the serum samples of HCC patients (p < 0.01). Additionally, the change of NA3Fb was not influenced by Hepatitis B virus (HBV)and cirrhosis. Furthermore, the mRNA and protein expression of N-acetylglucosaminyltransferase IVa (GnT-IVa), which was related to the synthesis of the NA3Fb, was substantially increased in HCC tissues. Knockdown of GnT-IVa leads to a decreased level of NA3Fb and decreased ability of invasion and migration in HCC cells. NA3Fb can be regarded as a specific cell surface N-glycan of HCC. The high expression of GnT-IVa is the cause of the abnormal increase of NA3Fb on the HCC cell surface, which regulates cell migration. This study demonstrated the specific N-glycans of the cell surface and the mechanisms of altered glycoform related with HCC. These findings lead to better understanding of the function of glycan and glycosyltransferase in the tumorigenesis, progression and metastasis of HCC.

MARVELD1 inhibited cell proliferation and enhance chemosensitivity via increasing expression of p53 and p16 in hepatocellular carcinoma

We have previously found that expression of MARVELD1 was remarkably downregulated in multiple tumor tissues, but unclear in hepatocellular carcinoma (HCC) and its function has not been explored yet. In the present study, to uncover the underlying mechanism of MARVELD1 in the pathogenesis and development of HCC, we investigated the expression pattern of MARVELD1 and its effect on tumor proliferation in HCC. The results indicated the frequent downregulation of MARVELD1 in clinic samples and cell lines of HCC resulted from promoter methylation, as well as genetic deletion. Furthermore, treatment of MARVELD1 unexpressing Hep3B2.1‐7 and PLC/PRF/5 cells with the demethylating agent 5‐aza‐2′ deoxycytidine restored its expression. Overexpression of MARVELD1 suppressed the proliferation of HCC cells in vitro and in vivo, whereas downregulation of endogenous MARVELD1 by shRNAs significantly enhanced these characters. MARVELD1 overexpression could enhance chemosensitivity of HCC cells to epirubicin and 10‐hydroxycamptothecin. Corresponding to these results, the expression of p‐ERK1/2 and cyclin D1 were decreased, whereas p16 and p53 were increased in MARVELD1‐transfected cells. We also demonstrated that knockdown of MARVELD1 resulted in upregulation of p‐ERK1/2 and cyclin D1, and downregulation of p16 and p53. Moreover, the effect of the decreased cell growth rate was significantly reversed when MARVELD1‐overexpressing cells were trasfected with p53 or p16 siRNA. Our findings suggest that MARVELD1 is a tumor suppressor by negatively regulating proliferation, tumor growth and chemosensitivity of HCC cells via increasing p53 and p16 in vitro and in vivo. MARVELD1 may be a potential target for HCC therapy. (Cancer Sci 2012; 103: 716–722)

Biological and clinical significance of epigenetic silencing of MARVELD1 gene in lung cancer

Epigenetic silence in cancer frequently altered signal-transduction pathways during the early stages of tumor development. Recent progress in the field of cancer epigenetics has led to new opportunities for diagnosis and treatment of cancer. We previously demonstrated that novel identified nuclear factor MARVELD1 was widely expressed in human tissues, but down-regulated by promoter methylation in multiple cancers. This study was carried out to determine the biological and clinical significance of MARVELD1 gene silencing in lung cancer. Here, we found the reduced MARVELD1 expression significantly correlated with diagnostic histopathology and malignant degree of lung cancers. DNA hypermethylation and histone deacetylation synergistically inactivated MARVELD1 gene in lung cancer cells. Moreover, MARVELD1 modulated the efficiency of nonsense-mediated mRNA decay (NMD) through interaction with NMD core factor SMG1. The decreased MARVELD1 level in lung cancer reduces NMD efficiency through diminishing the association between NMD complex component UPF1/SMG1 and premature termination codons containing mRNA (PTC-mRNA). The results suggested that MARVELD1 silencing is an appealing diagnostic biomarker for lung cancer and epigenetic silencing of MARVELD1 gene links with the regulatory mechanism of NMD pathway in lung cancer, which may be required for tumorigenesis.

Sialylation and desialylation dynamics of monocytes upon differentiation and polarization to macrophages

Sialic acids (SAs) often exist as the terminal sugars of glycan structures of cell surface glycoproteins and glycolipids. The level and linkages of cell surface SAs, which are controlled by both sialylation and desialylation processes and environment cues, can dramatically impact cell properties and represent different cellular status. In this study, we systematically examined the sialylation and desialylation profiles of THP-1 monocytes after differentiation to M0 macrophages, and polarization to M1 and M2 macrophages by the combination of LC-MS/MS, flow cytometry and confocal microscopy. Interestingly, both α2-3- and α2-6-linked SAs on the cell surface decreased after monocytes were differentiated to macrophages, which was in accordance with the increased level of free SA in the cell culture medium and the elevated activity of endogenous Neu1 sialidase. Meanwhile, the siaoglycoconjugates inside the cells increased as confirmed by confocal microscopy and the total SA inside the cells increased as determined by LC-MS/MS. Western blot analysis showed higher expression levels of sialyltransferases, including ST3Gal-I, ST3Gal-V, ST6Gal-I and ST6GalNAc-II. Further, upon polarization, the cell surface sialylation levels of M1 and M2 macrophages remained the same as M0 macrophages, while a slight decrease of cellular SAs in the M1 macrophages but increase in the M2 macrophages were confirmed by LC-MS/MS.