We have shown that glycosylation of influenza A virus (IAV) hemagglutinin (HA), especially at position N-27, is crucial for HA folding and virus survival. However, it is not known whether the glycosylation of HA and the other two major IAV surface glycoproteins, neuraminidase (NA) and M2 ion channel, is essential for the replication of IAV. Here, we show that glycosylation of HA at N-142 modulates virus infectivity and host immune response. Glycosylation of NA in the stalk region affects its structure, activity, and specificity, thereby modulating virus release and virulence, and glycosylation at the catalytic domain affects its thermostability; however, glycosylation of M2 had no effect on its function. In addition, using IAV without the stalk and catalytic domains of NA as a live attenuated vaccine was shown to confer a strong IAVspecific CD8+ T-cell response and a strong cross-strain as well as cross-subtype protection against various virus strains.influenza A virus | glycosylation | vaccine design I nfluenza A viruses (IAV) belong to the Orthomyxoviridae family and can circulate widely and cross interspecies barriers through the highly antigenic drift and shift of the 18 subtypes of HA and 11 subtypes of neuraminidase (NA) (1, 2). In addition, the posttranslational modification of the IAV surface proteins is important to circumvent host defense and support the virus life cycle (3).HA is a major surface glycoprotein of IAV and is involved in viral infection via binding to sialic acid (SA)-containing glycans on the surface of host cell (3). The other major glycoprotein of IAV, NA, is involved in the cleavage of SA on the host cell receptor to facilitate the release of viral particles to infect other cells (4). M2, the third surface protein of IAV, has ion channel activity to regulate virus penetration and uncoating (1). All surface proteins interact with M1 protein for virus assembly and release (5).The modification of HA and NA by N-glycosylation is important in the IAV life cycle (1, 6-8). Previously, we have shown that the glycosylation of HA affects its receptor binding, immune response, and structural stability, and glycosite 27 is essential for retaining the structural integrity of HA and its receptor binding (6, 9). In addition, using the monoglycosylated HA as immunogen, it showed a broader protection against various IAV subtypes compared with the fully glycosylated version (10). However, it is not clear whether the other specific glycosites and their glycan structures on HA regulate its functions. With regard to NA, the functional roles of its glycosylation are not well understood, though N-glycosylation was reported to stabilize the protein from protease digestion and may affect the enzyme activity (11,12). The aim of this study was to understand the functional effects of glycosylation on HA, NA, and M2, and how glycosylation of the surface proteins affects the life cycle of IAV. ResultsGlycosylation of HA at N-142. Because several glycosylation sites (glycosites 27, 40, 176, 303, and 497) on HA are ...

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme β1,3-galactosyltransferase V (β3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of β3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of β3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.

The importance of epigenetic dysregulation to acute myeloid leukemia (AML) pathophysiology has become increasingly apparent in recent years. Epigenetic regulators, including readers, writers, and erasers, are recurrently dysregulated by way of chromosomal translocations, somatic mutations, or genomic amplification in AML and many of these alterations are directly implicated in AML pathogenesis. Mutations in epigenetic regulators are often discovered in founder clones and persist after therapy, indicating that they may contribute to a premalignant state poised for the acquisition of cooperating mutations and frank malignancy. Apart from the proto-oncogenic impact of these mutations, the AML epigenome is also shaped by other epigenetic factors that are not mutated but co-opted by AML oncogenes, presenting with actionable vulnerabilities in this disease. Targeting the AML epigenome might also be important for eradicating AML leukemia stem cells, which can be critical for disease maintenance and resistance to therapy. In this review, we describe the importance of epigenetic regulators in AML. We also summarize evidence implicating specific epigenetic regulators in AML pathobiology and discuss emerging epigenome-based therapies for the treatment of AML in the clinic.

Quiescent cells are considered to be dormant. However, recent studies suggest that quiescent fibroblasts possess active metabolic profile and certain functional characteristics. We previously observed that serum-starved quiescent fibroblasts respond to proinflammatory stimuli by robust expression of cyclooxygenase-2 (COX-2), which declines after the quiescent fibroblasts are driven to proliferation. In this study, we elucidated the underlying signaling and transcriptional mechanism and identified by microarray genes with similar differential expression. By using pharmacological inhibitors coupled with gene silencing, we uncovered the key role of protein kinase C δ (PKCδ) and extracellular signal regulated protein kinase 1/2 (ERK1/2) signaling in mediating COX-2 expression in quiescent cells. Surprisingly, COX-2 expression in proliferative cells was not blocked by PKCδ or ERK1/2 inhibitors due to intrinsic inhibition of PKCδ and ERK1/2 in proliferative cells. Restrained COX-2 transcription in proliferative cells was attributable to reduced NF-κB binding. Microarray analysis identified 35 genes whose expressions were more robust in quiescent than in proliferative cells. A majority of those genes belong to proinflammatory cytokines, chemokines, adhesive molecules and metalloproteinases, which require NF-κB for transcription. Quiescent fibroblasts had a higher migratory activity than proliferative fibroblasts as determined by the transwell assay. Selective COX-2 inhibition reduced migration which was restored by prostaglandin E2. As COX-2 and inflammatory mediators induce DNA oxidation, we measured 8-hydroxydeoxyguanosine (8-OHdG) in quiescent vs. proliferative fibroblasts. PMA-induced 8-OHdG accumulation was significantly higher in quiescent than in proliferative fibroblasts. These findings indicate that quiescent fibroblasts (and probably other quiescent cells) are at the forefront in mounting inflammatory responses through expression of an array of proinflammatory genes via the PKCδ/ERK1/2 signaling pathway.