N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global public health crisis. The causative agent, the SARS-CoV-2 virus, enters host cells via molecular interactions between the viral spike protein and the host cell ACE2 surface protein. The SARS-CoV-2 spike protein is extensively decorated with up to 66 N-linked glycans. Glycosylation of viral proteins is known to function in immune evasion strategies but may also function in the molecular events of viral entry into host cells. Here, we show that N-glycosylation at Asn331 and Asn343 of SARS-CoV-2 spike protein is required for it to bind to ACE2 and for the entry of pseudovirus harboring the SARS-CoV-2 spike protein into cells. Interestingly, high-content glycan binding screening data have shown that N-glycosylation of Asn331 and Asn343 of the RBD is important for binding to the specific glycan molecule G4GN (Galß-1,4 GlcNAc), which is critical for spike-RBD-ACE2 binding. Furthermore, IL-6 was identified through antibody array analysis of conditioned media of the corresponding pseudovirus assay. Mutation of N-glycosylation of Asn331 and Asn343 sites of the spike receptor-binding domain (RBD) significantly reduced the transcriptional upregulation of pro-inflammatory signaling molecule IL-6. In addition, IL-6 levels correlated with spike protein levels in COVID-19 patients' serum. These findings establish the importance of RBD glycosylation in SARS-CoV-2 pathogenesis, which can be exploited for the development of novel therapeutics for COVID-19.

A Case-Control Study of Follicular Fluid Cytokine Profiles in Women with Diminished Ovarian Reserve.

Ovarian reserve is an important determinant of a woman's reproductive potential, and women with diminished ovarian reserve (DOR) often seek in vitro fertilization (IVF). The underlying etiology of DOR is unknown, but follicular fluid cytokine concentrations likely play a role in follicular development and maturation. The present study seeks to investigate the expression of cytokines in follicular fluid (FF) of women with DOR undergoing IVF and explore correlated functional pathways. One hundred ninety-four women undergoing ovarian stimulation were recruited at the time of oocyte retrieval. Women were classified as having DOR if they met one or more of the following criteria: AMH < 1 ng/ml, FSH > 10 mIU/ml, and/or AFC < 10. Controls included women undergoing IVF for male factor, tubal factor due to tubal ligation, or planned oocyte cryopreservation (non-oncologic). The concentrations of 480 cytokines and related growth factors in follicular fluid were determined using a multiplex immunoassay. Fifty-nine cytokines had significantly different concentrations (53 higher and 6 lower) in the DOR relative to the control group after adjusting for age and body mass index (BMI) (false discovery rate; FDR < 0.1). Using the most informative 44 biomarkers as indicated by a random forest (RF) model, an area under the curve (AUC) of 0.78 was obtained. Thus, follicular microenvironment differs between women with DOR and normal ovarian reserve. The differentially expressed cytokines belong to diverse processes that are primarily involved in follicular maturation and ovulation. These changes may play an important role in treatment outcomes in women with DOR.

Decreased consumption of rewarding sucrose solutions after injection of melanocortins into the ventral tegmental area of rats.

RATIONALE: The mesolimbic dopamine system is an important component of the neural circuitry controlling reward-related behavior. We have recently shown that the melanocortin peptides decrease normal homeostatic feeding through actions in the ventral tegmental area. It is unknown, however, whether melanocortin peptides can also act on dopamine pathways to regulate hedonic, reward-related aspects of feeding. OBJECTIVES: In these studies, we tested whether injection of melanocortin receptor agonists directly into the ventral tegmental area (VTA) affected the intake of appetizing and rewarding sugar solutions in two-bottle choice tests. METHODS: Varying doses of the melanocortin receptor agonist, MTII, were injected into the VTA, and the intake of different sugar solutions was measured in two-bottle choice tests to distinguish between potential effects on homeostatic versus hedonic aspects of feeding. In addition, 24-h food intake was measured throughout the experiments. RESULTS: Injection of MTII into the VTA dose dependently decreased the intake of 1 and 2 % sucrose solutions and 0.2 % saccharin solutions and decreased 24-h food intake in each study. Although MTII also decreased the intake of a 10 % sucrose solution, MTII appeared to be less potent in rats exposed to 10 % sucrose, as only the highest dose of MTII tested was effective at reducing 10 % sucrose intake and food intake in these rats. CONCLUSIONS: These studies demonstrate that melanocortins can act directly in the VTA to control reward-related feeding. Thus, these studies add to the growing body of evidence showing that melanocortins can interact with the mesolimbic dopamine system to regulate multiple reward-related behaviors.