External control of gene expression is crucial in synthetic biology and biotechnology research and applications, and is commonly achieved using inducible promoter systems. The E. coli rhamnose-inducible rhaBAD promoter has properties superior to more commonly used inducible expression systems, but is marred by transient expression caused by degradation of the native inducer, l-rhamnose. To address this problem, 35 analogues of l-rhamnose were screened for induction of the rhaBAD promoter, but no strong inducers were identified. In the native configuration, an inducer must bind and activate two transcriptional activators, RhaR and RhaS. Therefore, the expression system was reconfigured to decouple the rhaBAD promoter from the native rhaSR regulatory cascade so that candidate inducers need only activate the terminal transcription factor RhaS. Rescreening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterized further to determine the strength, kinetics, and concentration-dependence of induction; whether the inducer was used as a carbon source by E. coli; and the modality (distribution) of induction among populations of cells. l-Mannose was found to be the most useful orthogonal inducer, providing an even greater range of induction than the native inducer l-rhamnose, and crucially, allowing sustained induction instead of transient induction. These findings address the key limitation of the rhaBAD expression system and suggest it may now be the most suitable system for many applications.

Background Nucleic acids are potent stimulators of type I interferon (IFN-I) and antiviral defense, but may also promote pathological inflammation. A range of diseases are characterized by elevated IFN-I, including systemic lupus erythematosus (lupus). The DNA-activated cGAS-STING pathway is a major IFN-I-inducing pathway, and activation of signaling is dependent on trafficking of STING from the ER to the Golgi. Methods Here we used cell culture systems, a mouse lupus model, and material from lupus patients, to explore the mode of action of a STING antagonistic peptide, and its ability to modulate disease processes. Findings We report that the peptide ISD017 selectively inhibits all known down-stream activities of STING, including IFN-I, inflammatory cytokines, autophagy, and apoptosis. ISD017 blocks the essential trafficking of STING from the ER to Golgi through a mechanism dependent on the STING ER retention factor STIM1. Importantly, ISD017 blocks STING activity in vivo and ameliorates disease development in a mouse model for lupus. Finally, ISD017 treatment blocks pathological cytokine responses in cells from lupus patients with elevated IFN-I levels. Interpretation These data hold promise for beneficial use of STING-targeting therapy in lupus. Funding The Novo Nordisk Foundation, The European Research Council, The Lundbeck Foundation, European Union under the Horizon 2020 Research, Deutsche Forschungsgemeinschaft, Chulalongkorn University.

Herein we report on the development of novel glycosylation methodology for the concise synthesis of naturally occurring glycoconjugate motifs containing N-acetylgalactosamine (GalNAc) from the cheaper and commercially available N-acetylglucosamine (GlcNAc). The stereoselective glycosylations proceed with catalytic amounts of a promoter and without the need for N-protection other than the biologically relevant N-acetyl group. Among the catalysts explored, both Bi(OTf)3 and Fe(OTf)3 were found to be highly active Lewis acids for this reaction. It was also found that other less reactive metal triflates such as those of Cu(II) and Yb(III) can be beneficial in glycosylation reactions on more demanding glycosyl acceptors. We have furthermore demonstrated that it is possible to control the anomeric stereoselectivity in the glycosylation via postglycosylation in situ anomerization to obtain good yields of α-galactosides. The present protocol was used to prepare important naturally occurring carbohydrate motifs, including a trisaccharide fragment of the naturally occurring marine sponge clarhamnoside.

The monosaccharide N-acetyl-d-glucosamine (GlcNAc) is an abundant building block in naturally occurring oligosaccharides, but its incorporation by chemical glycosylation is challenging since direct reactions are low yielding. This issue, generally agreed upon to be caused by an intermediate 1,2-oxazoline, is often bypassed by introducing extra synthetic steps to avoid the presence of the NHAc functional group during glycosylation. The present paper describes new fundamental mechanistic insights into the inherent challenges of performing direct glycosylation with GlcNAc. These results show that controlling the balance of oxazoline formation and glycosylation is key to achieving acceptable chemical yields. By applying this line of reasoning to direct glycosylation with a traditional thioglycoside donor of GlcNAc, which otherwise affords poor glycosylation yields, one may obtain useful glycosylation results.

Multiple sclerosis (MS) is an autoimmune disorder manifested via chronic inflammation, demyelination, and neurodegeneration inside the central nervous system. The progressive phase of MS is characterized by neurodegeneration, but unlike classical neurodegenerative diseases, amyloid-like aggregation of self-proteins has not been documented. There is evidence that citrullination protects an immunodominant peptide of human myelin oligodendrocyte glycoprotein (MOG34–56) against destructive processing in Epstein-Barr virus-infected B-lymphocytes (EBV-BLCs) in marmosets and causes exacerbation of ongoing MS-like encephalopathies in mice. Here we collected evidence that citrullination of MOG can also lead to amyloid-like behavior shifting the disease pathogenesis toward neurodegeneration. We observed that an immunodominant MOG peptide, MOG35–55, displays amyloid-like behavior upon site-specific citrullination at positions 41, 46, and/or 52. These amyloid aggregates are shown to be toxic to the EBV-BLCs and to dendritic cells at concentrations favored for antigen presentation, suggesting a role of amyloid-like aggregation in the pathogenesis of progressive MS.