The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells have proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here, we report on the creation of fluorescent uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT, we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time- and dose-dependent manner. Because no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular physiology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-color pulse chase experiments and monitoring OGT activity on specific protein substrates in live cells.

O-glycosylation of the nuclear pore complex (NPC) by O-linked N-acetylglucosamine (O-GlcNAc) is conserved within metazoans. Many nucleoporins (Nups) comprising the NPC are constitutively O-GlcNAcylated, but the functional role of this modification remains enigmatic. We show that loss of O-GlcNAc, induced by either inhibition of O-GlcNAc transferase (OGT) or deletion of the gene encoding OGT, leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups. Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation. The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells. These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter. The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycosylation of the NPC among metazoans.

ObjectiveHCV prevails in uremic haemodialysis patients. The current study aimed to achieve HCV microelimination in haemodialysis centres through a comprehensive outreach programme.DesignThe ERASE-C Campaign is an outreach programme for the screening, diagnosis and group treatment of HCV encompassing 2323 uremic patients and 353 medical staff members from 18 haemodialysis centres. HCV-viremic subjects were linked to care for directly acting antiviral therapy or received on-site sofosbuvir/velpatasvir therapy. The objectives were HCV microelimination (>80% reduction of the HCV-viremic rate 24 weeks after the end of the campaign in centres with ≥90% of the HCV-viremic patients treated) and ‘No-C HD’ (no HCV-viremic subjects at the end of follow-up).ResultsAt the preinterventional screening, 178 (7.7%) uremic patients and 2 (0.6%) staff members were HCV-viremic. Among them, 146 (83.9%) uremic patients received anti-HCV therapy (41 link-to-care; 105 on-site sofosbuvir/velpatasvir). The rates of sustained virological response (SVR12, undetectable HCV RNA 12 weeks after the end of treatment) in the full analysis set and per-protocol population were 89.5% (94/105) and 100% (86/86), respectively, in the on-site treatment group, which were comparable with the rates of 92.7% (38/41) and 100% (38/38), respectively, in the link-to-care group. Eventually, the HCV-viremic rate decreased to 0.9% (18/1,953), yielding an 88.3% reduction from baseline. HCV microelimination and ‘No-C HD’ were achieved in 92.3% (12/13) and 38.9% (7/18) of the haemodialysis centres, respectively.ConclusionOutreach strategies with mass screenings and on-site group treatment greatly facilitated HCV microelimination in the haemodialysis population.ClinicalTrials.gov identifierNCT03803410 and NCT03891550

Background and aimsChronic hepatitis B patients in Taiwan with no or limited liver injury are not reimbursed for antiviral treatment by the Taiwan National Health Insurance (NHI). Innovative fibrosis marker, age-adjusted Fibrosis-4 Index (FIB4-AA), was implemented to evaluate the tendency of liver fibrosis in these patients.MethodsThe FIB-4 indices of 256 antiviral treatment-naïve chronic hepatitis B patients at Kaohsiung Medical University Hospital from 2003 to 2019 were reviewed. The difference in initial FIB-4 and last FIB4-AA was treated as a categorical variable, representing the tendency of liver fibrosis in each individual aside from ageing. Logistic regression was implemented to evaluate the three parameters most dependent on increment of FIB4-AA: e seroconversion, body mass index (BMI) and initial FIB-4 index.ResultsThe yearly FIB-4 growth rate of an individual without chronic hepatitis was lower than that of the study group (0.0237 vs 0.0273 for males, 0.02 vs 0.0288 for females). Patients undergoing or completing e seroconversion were less prone to increment of FIB4-AA (p=0.036, OR 0.524). Logistic regression revealed that BMI ≥25 kg/m2 significantly less increment of FIB4-AA (p=0.001, OR 0.383, 95% CI 0.212 to 0.690), while patients with initial FIB-4 <1.29 were prone to increasing liver FIB4-AA (p=0.000, OR 3.687, 95% CI 1.999 to 6.797).ConclusionChronic hepatitis B patients not meeting the reimbursement criteria of the Taiwan NHI are prone to increment of liver fibrosis marker. Overweight is associated with less increment of fibrosis marker, while initial FIB-4 <1.29 is associated with increasing fibrosis marker.