Cystathionine β‐synthase (CBS) deficiency, a genetic disorder in homocysteine (Hcy) metabolism in humans, elevates plasma Hcy‐thiolactone and leads to connective tissue abnormalities that affect the cardiovascular and skeletal systems. However, the underlying mechanism of these abnormalities is not understood. Hcy‐thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates N ‐homocysteinylated protein. Because lysine residues are involved in collagen cross‐linking, N ‐homocysteinylation of these lysines should impair cross‐linking. Using a Tg‐I278T Cbs ^−/− mousemodel of hyperhomocysteinemia(HHcy)which replicates the connective tissue abnormalities observed in CBS‐deficient patients, we found that N ‐Hcy‐collagen was elevated in bone, tail, and heartof Cbs ^−/− mice, whereas pyridinoline cross‐links were significantly reduced. Plasma deoxypyridinoline cross‐link and cross‐linked carboxyterminal telopeptide of type I collagen were also significantly reduced in the Cbs ^−/− mice. Lysine oxidase activity and mRNA level were not reduced by the Cbs ^−/− genotype. We also showed that collagen carries S‐linked Hcy bound to the thiol of N ‐linked Hcy. in vitro experiments showed that Hcy‐thiolactone modifies lysine residues in collagen type I α‐1 chain. Residue K ¹⁶⁰ , located in the nonhelical N ‐telopeptide region and involved in pyridinoline cross‐link formation, was also N ‐homocysteinylated in vivo . Taken together, our findings showed that N ‐homocysteinylation of collagen in Cbs ^−/− mice impairs its cross‐linking. These findings explain, at least in part, connective tissue abnormalities observed in HHcy.—Perla‐Kajan, J., Utyro, O., Rusek, M., Malinowska, A., Sitkiewicz, E., Jakubowski, H. N ‐Homocysteinylation impairs collagen cross‐linking in cystathionine β‐synthase‐deficient mice: a novel mechanism of connective tissue abnormalities. FASEB J. 30, 3810–3821 (2016) www.fasebj.org

Recent evidence suggests that SARS-CoV-2 hinders immune responses via dopamine (DA)-related mechanisms. Nonetheless, studies addressing the specific role of DA in the frame of SARS-CoV-2 infection are still missing. In the present study, we investigate the role of DA in SARS-CoV-2 replication along with potential links with innate immune pathways in CaLu-3 human epithelial lung cells. We document here for the first time that, besides DA synthetic pathways, SARS-CoV-2 alters the expression of D1 and D2 DA receptors (D1DR, D2DR), while DA administration reduces viral replication. Such an effect occurs at non-toxic, micromolar-range DA doses, which are known to induce receptor desensitization and downregulation. Indeed, the antiviral effects of DA were associated with a robust downregulation of D2DRs both at mRNA and protein levels, while the amount of D1DRs was not significantly affected. While halting SARS-CoV-2 replication, DA, similar to the D2DR agonist quinpirole, upregulates the expression of ISGs and Type-I IFNs, which goes along with the downregulation of various pro-inflammatory mediators. In turn, administration of Type-I IFNs, while dramatically reducing SARS-CoV-2 replication, converges in downregulating D2DRs expression. Besides configuring the CaLu-3 cell line as a suitable model to study SARS-CoV-2-induced alterations at the level of the DA system in the periphery, our findings disclose a previously unappreciated correlation between DA pathways and Type-I IFN response, which may be disrupted by SARS-CoV-2 for host cell invasion and replication.

The BNT162b2 vaccine induces neutralizing activity (NA) in serum, but no data are available on whether a third-dose activates specific-immunity within the oral mucosa, representing the primary route of viral-entry. To carefully address this issue, we investigated if such immunity is boosted by SARS-CoV-2-infection; how long it is maintained over-time; and if it protects against the SARS-CoV-2 lineage B.1 (EU) and the emerging Delta and Omicron variants. NA was measured in plasma and saliva samples from: uninfected SARS-CoV-2-Vaccinated (SV), subjects infected prior to vaccination (SIV), and subjects who were infected after the second (SIV2) or the third (SIV3) vaccine dose. Samples were collected immediately before (T0), 15 days (T1), and 90 days (T2) post third-dose administration (SV and SIV), or 15 days post-infection (SIV2 and SIV3). In all the enrolled groups, NA in plasma and saliva: (i) was higher against EU compared to the other variants at all time-points (SV: T0 and T1, EU vs. both Delta and Omicron p < 0.001; T2 p < 0.01) (SIV: T0, EU vs. Delta p < 0.05; EU vs. Omi p < 0.01; T1 and T2 EU vs. Delta p < 0.01; EU vs. Omi p < 0.001); (ii) was boosted by the administration of the third dose; iii) declined over-time, albeit being detectable in almost all subjects at T2. The monitoring of NA over time will be important in clarifying if different NA levels may influence either acquisition or course of infection to properly plan the timing of a fourth vaccine dose administration.