Publications - Publikationer

Exploiting O-GlcNAc dyshomeostasis to screen O-GlcNAc transferase intellectual disability variants

Yuan, H., Mitchell, C. W., Ferenbach, A. T., et al. — Tue, 14 Jan 2025 10:20:30 +0100

O-GlcNAcylation is an essential protein modification catalyzed by O-GlcNAc transferase (OGT). Missense variants in OGT are linked to a novel intellectual disability syndrome known as OGT congenital disorder of glycosylation (OGT-CDG). The mechanisms by which OGT missense variants lead to this heterogeneous syndrome are not understood, and no unified method exists for dissecting pathogenic from non-pathogenic variants. Here, we develop a double-fluorescence strategy in mouse embryonic stem cells to measure disruption of O-GlcNAc homeostasis by quantifying the effects of variants on endogenous OGT expression. OGT-CDG variants generally elicited a lower feedback response than wild-type and Genome Aggregation Database (gnomAD) OGT variants. This approach was then used to dissect new putative OGT-CDG variants from pathogenic background variants in other disease-associated genes. Our work enables the prediction of pathogenicity for rapidly emerging de novo OGT-CDG variants and points to reduced disruption of O-GlcNAc homeostasis as a common mechanism underpinning OGT-CDG.

Control of neonatal diarrhea in piglets with reduced antibiotic use by application of a complementary feed - a randomized controlled farm trial

Sall, K. K., Foldager, L., Delf, C., et al. — Fri, 10 Jan 2025 10:20:30 +0100

The objective of this farm trial was to investigate if the consumption of antibiotics could be reduced when piglets showing early signs of neonatal diarrhea were treated with an oral dose of tannin extract derived from sweet chestnut wood. The farm had a very high incidence of neonatal diarrhea among gilt litters. Gilts were randomized into test or control groups in a 1:1 ratio to compare the consumption of antibiotics used for piglets and piglet mortality during the four-week trial period. Control litters were treated with the oral antibiotic paromomycin, while test litters were treated with the complementary feed O-Nella-Protect. The farm trial included 18 gilt litters comprising 254 piglets. In the control group, 100% of the piglets received antibiotic treatment. In the test group, consumption of antibiotics used against diarrhea was reduced by 84% (p = 0.001) and consumption of antibiotics used for other illnesses was reduced by 45% (p = 0.045). In both test and control groups, six piglets died. Microbiological analysis identified both potential bacterial and viral pathogens. In conclusion, the farm trial indicates that even under the challenge of potentially serious bacterial and viral pathogens, a complimentary feed containing a tannin extract can support piglet health and reduce antibiotic consumption.

Resilience and Charge-Dependent Fibrillation of functional amyloid

Golan, N., Parizat, A., Tabachnikov, O., et al. — Sat, 01 Feb 2025 10:20:30 +0100

FapC and FapB are biofilm-associated amyloids involved in the virulence of Pseudomonas and other bacteria. We herein demonstrate their exceptional thermal and chemical resilience, suggesting that their biofilm structures might withstand standard sterilization, thereby contributing to the persistence of Pseudomonas aeruginosa infections. Our findings also underscore the impact of environmental factors on functional amyloid in Pseudomonas (Fap) proteins, suggesting that orthologs in different Pseudomonas strains adapt to specific environments and roles. Challenging previous assumptions about a simple nucleation role for FapB in promoting FapC aggregation, the study shows a significant influence of FapC on FapB aggregation. The interaction between these FapB and FapC is intricate: FapB stabilizes FapC fibrils, while FapC slows down FapB fibrillation but can still serve as a cross-seeding template. This complex interplay is the key to understanding their roles in bacterial biofilms. Furthermore, the study highlights distinct differences between Fap and Escherichia coli's CsgA (curli) amyloid, where CsgB assumes a simple unidirectional role in nucleating CsgA fibrillation, emphasizing the importance of a comprehensive understanding of various amyloid systems. This knowledge is vital for developing effective intervention strategies against bacterial infections and leveraging the unique properties of these amyloids in technological applications such as novel bionanomaterials or protective coatings.

Exercise, hormesis and ageing

Radak, Z., Rattan, S. I. S. — Sat, 01 Feb 2025 10:20:30 +0100

Dimethyl labeling of N-terminal amines allows unambiguous identification of protein crosslinks

Sat, 01 Feb 2025 10:20:30 +0100

Protein crosslinks induced through either deliberate enzymatic oxidation or reactive oxidants (oxidative eustress/distress), are associated with multiple human pathologies including atherosclerosis, Alzheimer's and Parkinson's diseases. In many cases, the nature of the crosslinks, their position(s) either within (intramolecular) or between (intermolecular) polypeptide chains, and concentrations are unclear. Although limited data are available from specific antibodies, detailed characterization of protein crosslinks is often performed by mass spectrometric analysis of peptides from proteolytic digestion. Such analyses are challenging due to the low concentration of these species, and the complexity of their fragment ion spectra when compared to non-crosslinked species. We hypothesized that highly efficient and specific chemical amine labeling of the two N-termini in crosslinked peptides (compared to the single N-terminus of linear peptides), using “light” and “heavy” isotope-labelled reagents would facilitate identification, validation and quantification of crosslinks. This method was compared to a previous enzyme-catalyzed ¹⁸O C-terminal carboxylate labeling approach. N-terminal amine dimethyl labeling is shown to have major advantages over the ¹⁸O-approach including high labeling yields (92–100 %) and well-defined mass spectrometric isotope distribution patterns. This approach has allowed identification of novel dityrosine crosslinks between pair of tyrosine (Tyr, Y) residues in photo-oxidized β-casein (Y195-Y195, Y195-Y208, Y208-Y208), and α-synuclein exposed to nitrosative stress (Y39-Y39, Y39-Y125, Y39-Y133, Y133-Y136). This approach is also applicable to disulfide bond mapping, with 15 of 17 disulfides in serum albumin readily detected. These data indicate that dimethyl labeling is a highly versatile and efficient approach for the site-specific identification of oxidation- and nitration-induced crosslinks in proteins.

Nanobodies' duo facilitates ultrasensitive serum HER-2/neu immunoassays via enhanced avidity interactions

Boonkaew, S., Teodori, L., Vendelbo, M. H., Kjems, J., Ferapontova, E. E. — Wed, 15 Jan 2025 10:20:30 +0100

BACKGROUND: Existing liquid biopsy assays for protein biomarkers of cancer are mostly based on antibodies (Ab) contributing unfavorably to their high cost. Easy to express and modify in vitro, nanobodies may be a cost-effective alternative to Ab.

RESULTS: We show that serum HER-2/neu, a biomarker and target of aggressive HER-2/neu(+) cancers, can be accurately detected in a 1.2 h electrochemical cellulase-linked sandwich nanobody/aptamer assay on magnetic beads. Using a single nanobody receptor, 2Rs15d or 2Rb17c, reduces immunoassay's sensitivity by 35%-26 %. A combination of two nanobodies as a duo-receptor recovers the sensitivity of the enzyme-linked nanobody/aptamer-sorbent assay (ELNASA) to 11.9 ± 2.8 μC fM-1, due to the avidity effects making the nanobodies-duo binding properties comparable to those of Ab. Down to 0.1 fM HER-2/neu was detected by ELNASA in serum samples, with no interference from other blood-circulating proteins. In a 30 healthy-volunteers trial, ELNASA more accurately than optical ELISA assayed serum HER-2/neu.

SIGNIFICANCE: ELNASA performance rivals that of ELISA, yet estimated to be at least 200 times cheaper, due to the lower cost of nanobodies production, and may be better suited for routine clinical analysis of HER-2/neu, particularly, in low- and middle-income settings with limited resources. The ELNASA approach is generic and may be adapted for specific and ultrasensitive analysis of other blood-circulating proteins.

Young rat microbiota extracts strongly inhibit fibrillation of α-synuclein and protect neuroblastoma cells and zebrafish against α-synuclein toxicity

Shiraz, M. G., Nielsen, J., Widmann, J., et al. — Wed, 01 Jan 2025 10:20:30 +0100

The clinical manifestations of Parkinson's disease (PD) are driven by aggregation of α-Synuclein (α-Syn) in the brain. However, there is increasing evidence that PD may be initiated in the gut and thence spread to the brain, eg, via the vagus nerve. Many studies link PD to changes in the gut microbiome, and bacterial amyloid has been shown to stimulate α-Syn aggregation. Yet, we are not aware of any studies reporting on a direct connection between microbiome components and α-Syn aggregation. Here, we report that soluble extract from the gut microbiome of the rats, particularly young rats transgenic for PD, shows a remarkably strong ability to inhibit in vitro α-Syn aggregation and keep it natively unfolded and monomeric. The active component(s) are heat-labile molecule(s) of around 30- to 100-kDa size, which are neither nucleic acid nor lipid. Proteomic analysis identified several proteins whose concentrations in different rat samples correlated with the samples’ anti-inhibitory activity, while a subsequent pull-down assay linked the protein chaperone DnaK with the inhibitory activity of young rat's microbiome, confirmed in subsequent in vitro assays. Remarkably, the microbiome extracts also protected neuroblastoma SH-SY5Y cells and zebrafish embryos against α-Syn toxicity. Our study sheds new light on the gut microbiome as a potential source of protection against PD and opens up for new microbiome-based therapeutic strategies.

Investigating the interactions between an industrial lipase and anionic (bio)surfactants

López Hernández, M., Otzen, D. E., Pedersen, J. S. — Sat, 01 Feb 2025 10:20:30 +0100

In laundry formulations, synergies between amphiphiles and other additives such as enzymes increase sustainability through a large decrease in energy consumption. However, traditional surfactants are derived from petroleum, requiring chemical modifications (sulfonation, ethoxylation, or esterification) and generating environmental pollution through toxicity and low degradability. Use of biosurfactants removes these issues. To provide a firmer basis for the use of biosurfactants, we report on the interactions between the industrial lipase LIPEX® and three common biosurfactants, rhamnolipids, sophorolipids, and surfactin. The model surfactant sodium dodecyl sulfate (SDS) is included in the study for comparison. A thorough characterization by Small-angle X-ray scattering (SAXS) provides valuable information on the enzyme's oligomerization and the surfactant micelles' ellipsoidal morphology. Additionally, the enzymatic activity and complex formation in different surfactant mixtures are studied using isothermal titration calorimetry, activity assays, and SAXS. SDS activates the enzyme while promoting a controlled association of monomers while the biosurfactants inhibit the enzyme, independent of their effects on its quaternary structure. Rhamnolipids and surfactin promote lipase dimerization while sophorolipids have no significant effect on lipase quaternary structure. Based on these data, we propose a partial replacement that allows the enzyme to retain enzymatic activity while improving the environmental footprint of the formulation.

Construction of Small Genes with Repetitive Elements Using Oligo Extension and Golden Gate Assembly

Nguyen, M. T.A., Andersen, E. S., Pothoulakis, G. — Wed, 01 Jan 2025 10:20:30 +0100

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences and results in synthesis failure or delays by DNA synthesis vendors. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden Gate-compatible overhangs. Then synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate assembly.