Proximity interactions of the ubiquitin ligase Mind bomb 1 reveal a role in regulation of epithelial polarity complex proteins.

MIB1 belongs to the RING domain containing family of E3 ubiquitin ligases. In vertebrates, MIB1 plays an essential role in activation of Notch signaling during development, through the ubiquitination and endocytosis of Notch ligands. More recently, Notch independent functions for MIB1 have been described in centriole homeostasis, dendritic spine outgrowth and directional cell migration. Here we use proximity-dependent biotin identification (BioID) to define the MIB1 interactome that included 163 high confidence interactions with polypeptides linked to centrosomes and cilia, endosomal trafficking, RNA and DNA processing, the ubiquitin system, and cell adhesion. Biochemical analysis identified several proteins within these groups including CCDC14 and EPS15 that were ubiquitinated but not degraded when co-expressed with MIB1. The MIB1 interactome included the epithelial cell polarity protein, EPB41L5. MIB1 binds to and ubiquitinates EPB41L5 resulting in its degradation. Furthermore, MIB1 ubiquitinates the EPB41L5-associated polarity protein CRB1, an important determinant of the apical membrane. In polarized cells, MIB1 localized to the lateral membrane with EPB41L5 and to the tight junction with CRB1, CRB3 and ZO1. Furthermore, over expression of MIB1 resulted in altered epithelial cell morphology and apical membrane expansion. These results support a role for MIB1 in regulation of polarized epithelial cell morphology.

Adaptive Mutations in Influenza A/California/07/2009 Enhance Polymerase Activity and Infectious Virion Production.

Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (also known as CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering three PB1 and PA mutations into the parental CA/07 strain, we demonstrated that these mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.

Taxonomic differences of gut microbiomes drive cellulolytic enzymatic potential within hind-gut fermenting mammals.

Host diet influences the diversity and metabolic activities of the gut microbiome. Previous studies have shown that the gut microbiome provides a wide array of enzymes that enable processing of diverse dietary components. Because the primary diet of the porcupine, Erethizon dorsatum, is lignified plant material, we reasoned that the porcupine microbiome would be replete with enzymes required to degrade lignocellulose. Here, we report on the bacterial composition in the porcupine microbiome using 16S rRNA sequencing and bioinformatics analysis. We extended this analysis to the microbiomes of 20 additional mammals located in Shubenacadie Wildlife Park (Nova Scotia, Canada), enabling the comparison of bacterial diversity amongst three mammalian taxonomic orders (Rodentia, Carnivora, and Artiodactyla). 16S rRNA sequencing was validated using metagenomic shotgun sequencing on selected herbivores (porcupine, beaver) and carnivores (coyote, Arctic wolf). In the microbiome, functionality is more conserved than bacterial composition, thus we mined microbiome data sets to identify conserved microbial functions across species in each order. We measured the relative gene abundances for cellobiose phosphorylase, endoglucanase, and beta-glucosidase to evaluate the cellulose-degrading potential of select mammals. The porcupine and beaver had higher proportions of genes encoding cellulose-degrading enzymes than the Artic wolf and coyote. These findings provide further evidence that gut microbiome diversity and metabolic capacity are influenced by host diet.

Moderate Exercise Has Limited but Distinguishable Effects on the Mouse Microbiome.

The gut microbiome is known to have a complex yet vital relationship with host health. While both exercise and the gut microbiome have been shown to impact human health independently, the direct effects of moderate exercise on the intestinal microbiota remain unclear. In this study, we compared gut microbial diversity and changes in inflammatory markers associated with exercise over an 8-week period in mice that performed either voluntary exercise (VE) (n = 10) or moderate forced exercise (FE) (n = 11) and mice that did not perform any exercise (n = 21). VE mice, but not FE mice, had increased food intake and lean body mass compared to sedentary mice. The levels of inflammatory markers associated with exercise were similar for mice in all three groups. Traditional microbial profiles comparing operational taxonomic units (OTUs) in samples (P > 0.1) and multivariate analysis of beta diversity via Adonis testing (P > 0.1) did not identify significantly altered taxonomic profiles in the voluntary or forced exercise group compared to the sedentary controls. However, a random forests machine learning model, which takes into account the relationships between bacteria in a community, classified voluntary exercisers and nonexercisers with 97% accuracy at 8 weeks. The top bacteria used by the model allowed us to identify known taxa (Bacteroides, S24-7, and Lactobacillus) and novel taxa (Rikenellaceae and Lachnospiraceae) associated with exercise. Although aerobic exercise in mice did not result in significant changes of abundance in gut microbes or in host inflammatory response, more sophisticated computational methods could identify some microbial shifts. More study is needed on the effects of various exercise intensities and their impact on the gut microbiome. IMPORTANCE The bacteria that live in our gut have a complex yet vital relationship with our health. Environmental factors that influence the gut microbiome are of great interest, as recent research demonstrates that these microbes, mostly bacteria, are important for normal host physiology. Diseases such as obesity, diabetes, inflammatory bowel disease, and colon cancer have also been linked to shifts in the microbiome. Exercise is known to have beneficial effects on these diseases; however, much less is known about its direct impact on the gut microbiome. Our results illustrate that exercise has a moderate but measurable effect on gut microbial communities in mice. These methods can be used to provide important insight into other factors affecting the microbiome and our health.