During the study period, all dogs remained on their pre-trial diets, and no dietary changes were performed as part of the here presented study. However, we have utilized qPCR assays targeting the microbiota on various phylogenetic levels and also targeting bacterial groups that are major bacterial groups in the canine intestine and that have been shown to be important in canine IBD. Furthermore, in the current study, only fecal samples were analyzed, and the potential impact of treatment on the composition of the small intestinal mucosa-associated microbiota may have been missed. Previous studies have revealed that dogs with IBD have significant differences in small intestinal microbiota compared to controls, and future studies should evaluate the effect of probiotics on the small intestinal microbiota of these dogs. Also, in this study we assessed the fecal microbiota 30 days after the discontinuation of therapy, and it is possible that a transient change in the fecal microbiota during the administration period may have remained undetected and/or changed during the 30 days post-treatment. It has been speculated that IBD is associated with a loss of intestinal barrier function, as multiple genes encoding for proteins responsible for maintenance of intestinal barrier function were down-regulated in dogs with IBD in a previous study. The observation that the expression and distribution of occludin and claudin-2 in the large intestine were not significantly different between dogs treated with VSL#3 and the non-IBD control dogs, but were significantly different compared to the D-CT group, suggests potential Gefitinib effects of VSL#3 on intestinal barrier function, warranting further studies. Similar changes in the distribution of claudin-2 expression have been observed in humans with active UC, where claudin-2 was detected at the surface epithelium. Although members within the PUS family do not exhibit extensive sequence homology, they share an enzymatic domain that presents a high degree of structural similarity. The active site is located in between the two lobes of the catalytic core. PUS enzymes are highly specific, capable of recognising their target uridine when embedded in a particular structural context, avoiding random uridine modification within RNA molecules. The hPus1p enzyme is no exception, although it appears to have a more relaxed sequence specificity compared to other pseudouridine synthases. The TruA family is the most divergent compared to the other families. The major sites of modification by the eukaryotic Pus1p enzyme are positions 27, 28, 34, and 36 within tRNAs. In addition, yeast Pus1 has been shown to modify U2 snRNA. A few 
years ago, the Steroid receptor RNA Activator, a ncRNA emanating from the SRA gene, was characterised as a target of Pus1p. Multiple sites within the SRA were shown to be subject to pseudouridine modification, although only U206 within the H7 element was identified unambiguously. Lastly, the hPus1p enzyme is involved in the metabolic syndrome causing mitochondrial myopathy and sideroblastic anemia. We have characterised the catalytic domain of the hPus1p protein biochemically and structurally. A Vismodegib Hedgehog inhibitor truncated protein has significant levels of activity towards a target tRNA and on the specific H7 element from the SRA, when compared to the fulllength hPus1p enzyme. We also measured the affinity of the truncated form of hPus1p.