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Global in-depth analysis of N-glycosylation, as the most complex post-translational modification of proteins, is requiring methods being as sensitive, selective and reliable as possible. Here, an enhanced strategy for N-glycomics is presented comprising optimized sample preparation yielding enhanced glycoprotein recovery and permethylation efficiency, isotopic labelling for data quality control and relative quantification, integration of new N-glycan libraries (human and mouse), newly developed R-scripts matching experimental MS1 data to theoretical N-glycan compositions and bundled sequencing algorithms for MS2-based structural identification to ultimately enhance the coverage and accuracy of N-glycans. With this strategy the numbers of identified N-glycans are more than doubled compared with previous studies, exemplified by etanercept (more than 3-fold) and chicken ovalbumin (more than 2-fold) at nanogram level. The power of this strategy and applicability to biological samples is further demonstrated by comparative N-glycomics of human acute promyelocytic leukemia cells before and after treatment with all-trans retinoic acid, showing that N-glycan biosynthesis is slowed down and 57 species are significantly altered in response to the treatment. This improved analytical platform enables deep and accurate N-glycomics for glycobiological research and biomarker discovery.

Host cells develop the OAS/RNase L (2-5 oligoadenylate synthetase/ribonuclease L) system to degrade cellular and viral RNA, and/or the OASL/RIG-I (2-5 oligoadenylate synthetase like/retinoic acid inducible protein I) system to enhance RIG-I-mediated IFN induction, thus providing the first line of defense against viral infection. The 2-5 oligoadenylate synthetase like (OASL) protein may activate the OAS/RNase L system using its typical OAS-like domain (OLD) or mimic the K63-linked pUb to enhance antiviral activity of the OASL/RIG-I system using its two tandem ubiquitin-like domains (UBLs). We first describe that divergent avian (duck and ostrich) OASL inhibit the replication of a broad range of RNA viruses by activating and magnifying the OAS/RNase L pathway in a UBL-dependent manner. This is in sharp contrast to mammalian enzymatic OASL, which activates and magnifies the OAS/RNase L pathway in a UBL-independent manner, similar to 2-5 oligoadenylate synthetase 1 (OAS1). We further show that both avian and mammalian OASL can reversibly exchange to activate and magnify the OAS/RNase L and OASL/RIG-I system by introducing only three key residues, suggesting that ancient OASL possess 2-5A activity and has functionally switched to the OASL/RIG-I pathway recently. Our findings indicate the molecular mechanisms involved in the switching of avian and mammalian OASL molecules to activate and enhance the OAS/RNase L and OASL/RIG-I pathways in response to infection by RNA viruses.

Vitamin A (all-trans-retinol; retinol) is an essential human nutrient and plays an important role in several biological functions. However, under certain circumstances, retinol treatment can cause free radical generation and induce oxidative stress. In this study, we investigated photocytotoxicity and photomutagenicity of retinol using L5178Y/Tk(+/-) mouse lymphoma cells concomitantly exposed to retinol and ultraviolet A (UVA) light. While the cells treated with retinol alone at the doses of 5 or 10microg/ml in the absence of light did not increase the mutant frequency (MF) in the Tk gene, the treatment of the cells with 1-4microg/ml retinol under UVA light (1.38mW/cm(2) for 30min) increased the MF in the Tk gene in a dose-responsive manner. To elucidate the underlying mechanism of action, we also examined the mutational types of the Tk mutants by determining their loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 on which the Tk gene is located. The mutational spectrum for the retinol+UVA treatment was significantly different from those of the control and UVA alone. More than 93% of the mutants from retinol+UVA treatment lost heterozygosity at the Tk1 locus and the major type (58%) of mutations was LOHs extending to D11Mit42, an alternation involving approximately 6cM of the chromosome, whereas the main type of mutations in the control was non-LOH mutations. These results suggest that retinol is mutagenic when exposed to UVA in mouse lymphoma cells through a clastogenic mode-of-action.

In addition to occupational exposures to acrylamide (AA), concerns about AA health risks for the general population have been recently raised due to the finding of AA in food. In this study, we evaluated the genotoxicity of AA and its metabolite glycidamide (GA) in L5178Y/ Tk+/− mouse lymphoma cells. The cells were treated with 2–18 mM of AA or 0.125–4 mM of GA for 4 h without metabolic activation. The DNA adducts, mutant frequencies and the types of mutations for the treated cells were examined. Within the dose range tested, GA induced DNA adducts of adenine and guanine [N3-(2-carbamoyl-2-hydroxyethyl)-adenine and N7-(2-carbamoyl-2-hydroxyethyl)-guanine] in a linear dose-dependent manner. The levels of guanine adducts were consistently about 60-fold higher across the dose range than those of adenine. In contrast, no GA-derived DNA adducts were found in the cells treated with any concentrations of AA, consistent with a lack of metabolic conversion of AA to GA. However, the mutant frequency was significantly increased by AA at concentrations of 12 mM and higher. GA was mutagenic starting with the 2 mM dose, suggesting that GA is much more mutagenic than AA. The mutant frequencies were increased with increasing concentrations of AA and GA, mainly due to an increase of proportion of small colony mutants. To elucidate the underlying mutagenic mechanism, we examined the loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 for mutants induced by AA or GA. Compared to GA induced mutations, AA induced more mutants whose LOH extended to D11Mit22 and D11Mit74, an alteration of DNA larger than half of the chromosome. Statistical analysis of the mutational spectra revealed a significant difference between the types of mutations induced by AA and GA treatments (P = 0.018). These results suggest that although both AA and GA generate mutations through a clastogenic mode of action in mouse lymphoma cells, GA induces mutations via a DNA adduct mechanism whereas AA induces mutations by a mechanism not involving the formation of GA adducts.