Engineering the sialome of mammalian cells with sialic acid mimetics.

Mammalian glycans show a diversity in sialic acid capping, constituting the sialome. Sialic acids can be extensively modified chemically, yielding sialic acid mimetics (SAMs). Here, we present a protocol for detecting and quantifying incorporative SAMs using microscopy and flow cytometry, respectively. We detail steps for linking SAMS to proteins with western blotting. Lastly, we detail procedures for incorporative or inhibitory SAMs and how SAMs can be used for the on-cell synthesis of high-affinity Siglec ligands. For complete details on the use and execution of this protocol, please refer to Büll et al.1 and Moons et al.2.

UV light-induced spatial loss of sialic acid capping using a photoactivatable sialyltransferase inhibitor.

Sialic acids cap glycans displayed on mammalian glycoproteins and glycolipids and mediate many glycan-receptor interactions. Sialoglycans play a role in diseases such as cancer and infections where they facilitate immune evasion and metastasis or serve as cellular receptors for viruses, respectively. Strategies that specifically interfere with cellular sialoglycan biosynthesis, such as sialic acid mimetics that act as metabolic sialyltransferase inhibitors, enable research into the diverse biological functions of sialoglycans. Sialylation inhibitors are also emerging as potential therapeutics for cancer, infection, and other diseases. However, sialoglycans serve many important biological functions and systemic inhibition of sialoglycan biosynthesis can have adverse effects. To enable local and inducible inhibition of sialylation, we have synthesized and characterized a caged sialyltransferase inhibitor that can be selectively activated with UV-light. A photolabile protecting group was conjugated to a known sialyltransferase inhibitor (P-SiaFNEtoc). This yielded a photoactivatable inhibitor, UV-SiaFNEtoc, that remained inactive in human cell cultures and was readily activated through radiation with 365 nm UV light. Direct and short radiation of a human embryonic kidney (HEK293) cell monolayer was well-tolerated and resulted in photoactivation of the inhibitor and subsequent spatial restricted synthesis of asialoglycans. The developed photocaged sialic acid mimetic holds the potential to locally hinder the synthesis of sialoglycans through focused treatment with UV light and may be applied to bypass the adverse effects related to systemic loss of sialylation.

Gas6/Axl Axis Activation Dampens the Inflammatory Response in Osteoarthritic Fibroblast-like Synoviocytes and Synovial Explants.

Osteoarthritis (OA) is the most prevalent joint disease, and it is characterized by cartilage degeneration, synovitis, and bone sclerosis, resulting in swelling, stiffness, and joint pain. TAM receptors (Tyro3, Axl, and Mer) play an important role in regulating immune responses, clearing apoptotic cells, and promoting tissue repair. Here, we investigated the anti-inflammatory effects of a TAM receptor ligand, i.e., growth arrest-specific gene 6 (Gas6), in synovial fibroblasts from OA patients. TAM receptor expression was determined in synovial tissue. Soluble Axl (sAxl), a decoy receptor for the ligand Gas6, showed concentrations 4.6 times higher than Gas6 in synovial fluid of OA patients. In OA fibroblast-like synoviocytes (OAFLS) exposed to inflammatory stimuli, the levels of sAxl in the supernatants were increased, while the expression of Gas6 was downregulated. In OAFLS under TLR4 stimulation by LPS (Escherichia coli lipopolysaccharide), the addition of exogenous Gas6 by Gas6-conditioned medium (Gas6-CM) reduced pro-inflammatory markers including IL-6, TNF-α, IL-1ß, CCL2, and CXCL8. Moreover, Gas6-CM downregulated IL-6, CCL2, and IL-1ß in LPS-stimulated OA synovial explants. Pharmacological inhibition of TAM receptors by a pan inhibitor (RU301) or by a selective Axl inhibitor (RU428) similarly abrogated Gas6-CM anti-inflammatory effects. Mechanistically, Gas6 effects were dependent on Axl activation, determined by Axl, STAT1, and STAT3 phosphorylation, and by the downstream induction of the suppressors of the cytokine signaling family (SOCS1 and SOCS3). Taken together, our results showed that Gas6 treatment dampens inflammatory markers of OAFLS and synovial explants derived from OA patients associated with SOCS1/3 production.

Bacterial Oncotraits Rather than Spatial Organization Are Associated with Dysplasia in Ulcerative Colitis.

BACKGROUND AND AIMS: Colonic bacterial biofilms are frequently present in ulcerative colitis [UC] and may increase dysplasia risk through pathogens expressing oncotraits. This prospective cohort study aimed to determine [1] the association of oncotraits and longitudinal biofilm presence with dysplasia risk in UC, and [2] the relation of bacterial composition with biofilms and dysplasia risk. METHODS: Faeces and left- and right-sided colonic biopsies were collected from 80 UC patients and 35 controls. Oncotraits [FadA of Fusobacterium, BFT of Bacteroides fragilis, colibactin [ClbB] and Intimin [Eae] of Escherichia coli] were assessed in faecal DNA with multiplex quantitative polymerase chain reaction [qPCR]. Biopsies were screened for biofilms [n = 873] with 16S rRNA fluorescent in situ hybridiation. Shotgun metagenomic sequencing [n = 265], and ki67-immunohistochemistry were performed. Associations were determined with a mixed-effects regression model. RESULTS: Biofilms were highly prevalent in UC patients [90.8%] with a median persistence of 3 years (interquartile range [IQR] 2-5 years). Biofilm-positive biopsies showed increased epithelial hypertrophy [p = 0.025] and a reduced Shannon diversity independent of disease status [p = 0.015], but were not significantly associated with dysplasia in UC: adjusted odds ratio [aOR] 1.45, 95% confidence interval [CI] 0.63-3.40. In contrast, ClbB independently associated with dysplasia [aOR 7.16, 95% CI 1.75-29.28], and FadA and Fusobacteriales were associated with a decreased dysplasia risk in UC [aOR 0.23, 95% CI 0.06-0.83, p <0.01]. CONCLUSIONS: Biofilms are a hallmark of UC; however, because of their high prevalence are a poor biomarker for dysplasia. In contrast, colibactin presence and FadA absence independently associate with dysplasia in UC and might therefore be valuable biomarkers for future risk stratification and intervention strategies.