Myxococcus xanthus cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways. In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization. Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient. The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM. Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction. The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers. As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions. Immunoblotting was used to localize important motility and signaling proteins within the protein peaks. CsgA, the C-signal-producing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g cm ⁻³ ). Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g cm ⁻³ ) and low buoyant density (1.169 to 1.171 g cm ⁻³ ). FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM. The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM. Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g cm ⁻³ ) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A.

Aims/hypothesis: Individual variation in plasmaN-glycosylation has mainly been studied in the context of diabetes complications, and its role in type 1 diabetes onset is largely unknown. Our aims were to undertake a detailed characterisation of the plasma and IgGN-glycomes in patients with recent onset type 1 diabetes, and to evaluate their discriminative potential in risk assessment.Methods: In the first part of the study, plasma and IgGN-glycans were chromatographically analysed in a study population from the DanDiabKids registry, comprising 1917 children and adolescents (0.6–19.1 years) who were newly diagnosed with type 1 diabetes. A follow-up study compared the results for 188 of these participants with those for their 244 unaffected siblings. Correlation ofN-glycan abundance with the levels and number of various autoantibodies (against IA-2, GAD, ZnT8R, ZnT8W), as well as with sex and age at diagnosis, were estimated by using general linear modelling. A disease predictive model was built using logistic mixed-model elastic net regression, and evaluated using a 10-fold cross-validation.Results: Our study showed that onset of type 1 diabetes was associated with an increase in the proportion of plasma and IgG high-mannose and bisecting GlcNAc structures, a decrease in monogalactosylation, and an increase in IgG disialylation. ZnT8R autoantibody levels were associated with higher IgG digalactosylated glycan with bisecting GlcNAc. Finally, an increase in the number of autoantibodies (which is a better predictor of progression to overt diabetes than the level of any individual antibody) was accompanied by a decrease in the proportions of some of the highly branched plasmaN-glycans. Models including age, sex andN-glycans yielded notable discriminative power between children with type 1 diabetes and their healthy siblings, with AUCs of 0.915 and 0.869 for addition of plasma and IgGN-glycans, respectively.Conclusions/interpretation: We definedN-glycan changes accompanying onset of type 1 diabetes, and developed a predictive model based onN-glycan profiles that could have valuable potential in risk assessment. Increasing the power of tests to identify individuals at risk of disease development would be a considerable asset for type 1 diabetes prevention trials.Graphical

S-adenosylhomocysteine hydrolase (AHCY) is thought to be located at the sites of ongoing AdoMet-dependent methylation, presumably in the cell nucleus. Endogenous AHCY is located both in cytoplasm and the nucleus. Little is known regarding mechanisms that drive its subcellular distribution, and even less is known on how mutations causing AHCY deficiency affect its intracellular dynamics. Using fluorescence microscopy and GFP-tagged AHCY constructs we show significant differences in the intensity ratio between nuclei and cytoplasm for mutant proteins when compared with wild type AHCY. Interestingly, nuclear export of AHCY is not affected by leptomycin B. Systematic deletions showed that AHCY has two regions, located at both sides of the protein, that contribute to its nuclear localization, implying the interaction with various proteins. In order to evaluate protein interactions in vivo we engaged in bimolecular fluorescence complementation (BiFC) based studies. We investigated previously assumed interaction with AHCY-like-1 protein (AHCYL1), a paralog of AHCY. Indeed, significant interaction between both proteins exists. Additionally, silencing AHCYL1 leads to moderate inhibition of nuclear export of endogenous AHCY.