Ludwig T, Worsch S, Heikenwalder M, Daniel H, Hauner H, Bader BL. Metabolic and immunomodulatory effects of n-3 fatty acids are different in mesenteric and epididymal adipose tissue of diet-induced obese mice.

Small proteins play essential roles in bacterial physiology and virulence, however, automated algorithms for genome annotation are often not yet able to accurately predict the corresponding genes. The accuracy and reliability of genome annotations, particularly for small open reading frames (sORFs), can be significantly improved by integrating protein evidence from experimental approaches. Here we present a highly optimized and flexible bioinformatics workflow for bacterial proteogenomics covering all steps from (i) generation of protein databases, (ii) database searches and (iii) peptide-to-genome mapping to (iv) visualization of results. We used the workflow to identify high quality peptide spectrum matches (PSMs) for small proteins (≤ 100 aa, SP100) in Staphylococcus aureus Newman. Protein extracts from S. aureus were subjected to different experimental workflows for protein digestion and prefractionation and measured with highly sensitive mass spectrometers. In total, 175 with up to 100 aa (SP100) were identified. Out of these 24 (ranging from 9 to 99 aa) were novel and not contained in the used genome annotation.144 SP100 are highly conserved and were found in at least 50% of the publicly available S. aureus genomes, while 127 are additionally conserved in other staphylococci. Almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids.

Diet-induced obesity in mice can be achieved through the use of diets with different macronutrient compositions and textures. We aimed at determining the contribution of macronutrient composition to obesity development and associated pathophysiological changes in mice. C57BL/6N mice were offered a control, a high-fat or a Western-style diet, either as pellet (H for hard) or with identical composition in powder form (S for soft), resulting in C-S, C-H, HF-H, HF-S, W-H and W-S groups, respectively. Body fat distribution, expression levels of selected target genes in adipose tissues, clinical chemistry and hormone concentration in the blood, as well as liver TAG content were measured. The most striking finding was that all mice fed the different powder diets developed obesity with similar weight gain, whereas among the mice fed the pellet diets, only those given the HF and W diets became obese. This allowed us to separate diet-specific effects from obesity-mediated effects. Irrespective of the food texture, the W diet induced a more severe hepatosteatosis and higher activities of serum transaminases compared with the two other diets. Adipose tissue gene expression analysis revealed that leptin and adiponectin levels were not affected by the dietary composition per se, whereas uncoupling protein 1 and 11b-hydroxysteroid dehydrogenase type 1 levels were decreased by both dietary composition and changes in body weight. In conclusion, diets differing in macronutrient composition elicit specific pathophysiological changes, independently of changes in body weight. A diet high in both fat and sugars seems to be more deleterious for the liver than a HF diet.

Background Congenital ISG15 deficiency is a rare autoinflammatory disorder that is driven by chronically elevated systemic interferon levels and predominantly affects central nervous system and skin. Methods and results We have developed induced pluripotent stem cell‐derived macrophages and endothelial cells as a model to study the cellular phenotype of ISG15 deficiency and identify novel treatments. ISG15 –/– macrophages exhibited the expected hyperinflammatory responses, but normal phagocytic function. In addition, they displayed a multifaceted pathological phenotype featuring increased apoptosis/pyroptosis, oxidative stress, glycolysis, and acylcarnitine levels, but decreased glutamine uptake, BCAT1 expression, branched chain amino acid catabolism, oxidative phosphorylation, β‐oxidation, and NAD(P)H‐dependent oxidoreductase activity. Furthermore, expression of genes involved in mitochondrial biogenesis and respiratory chain complexes II–V was diminished in ISG15 –/– cells. Defective mitochondrial respiration was restored by transduction with wild‐type ISG15, but only partially by a conjugation‐deficient variant, suggesting that some ISG15 functions in mitochondrial respiration require ISGylation to cellular targets. Treatment with itaconate, dimethyl‐itaconate, 4‐octyl‐itaconate, and the JAK1/2 inhibitor ruxolitinib ameliorated increased inflammation, propensity for cell death, and oxidative stress. Furthermore, the treatments greatly improved mitochondria‐related gene expression, BCAT1 levels, redox balance, and intracellular and extracellular ATP levels. However, efficacy differed among the compounds according to read‐out and cell type, suggesting that their effects on cellular targets are not identical. Indeed, only itaconates increased expression of anti‐oxidant genes NFE2L2, HMOX1 , and GPX7 , and dimethyl‐itaconate improved redox balance the most. Even though itaconate treatments normalized the elevated expression of interferon‐stimulated genes, ISG15 –/– macrophages maintained their reduced susceptibility to influenza virus infection. Conclusions These findings expand the cellular phenotype of human ISG15 deficiency and reveal the importance of ISG15 for regulating oxidative stress, branched chain amino acid metabolism, and mitochondrial function in humans. The results validate ruxolitinib as treatment for ISG15 deficiency and suggest itaconate‐based medications as additional therapeutics for this rare disorder.