Liver-specific DICER1 syndrome model mice develop cystic liver tumors with defective primary cilia.

DICER1 syndrome is a tumor predisposition syndrome caused by familial genetic mutations in DICER1. Pathogenic variants of DICER1 have been discovered in many rare cancers, including cystic liver tumors. However, the molecular mechanisms underlying liver lesions induced by these variants remain unclear. In the present study, we sought to gain a better understanding of the pathogenesis of these variants by generating a mouse model of liver-specific DICER1 syndrome. The mouse model developed bile duct hyperplasia with fibrosis, similar to congenital hepatic fibrosis, as well as cystic liver tumors resembling those in Caroli's syndrome, intrahepatic cholangiocarcinoma, and hepatocellular carcinoma. Interestingly, the mouse model of DICER1 syndrome showed abnormal formation of primary cilia in the bile duct epithelium, which is a known cause of bile duct hyperplasia and cyst formation. These results indicated that DICER1 mutations contribute to cystic liver tumors by inducing defective primary cilia. The mouse model generated in this study will be useful for elucidating the potential mechanisms of tumorigenesis induced by DICER1 variants and for obtaining a comprehensive understanding of DICER1 syndrome. © 2024 The Pathological Society of Great Britain and Ireland.

Motif-guided identification of a glycoside hydrolase family 1 α-L-arabinofuranosidase in Bifidobacterium adolescentis.

Members of glycoside hydrolase family 1 (GH1) cleave glycosidic linkages with a variety of physiological roles. Here we report a unique GH1 member encoded in the genome of Bifidobacterium adolescentis ATCC 15703. This enzyme, BAD0156, was identified from over 2,000 GH1 sequences accumulated in a database by a genome mining approach based on a motif sequence. A recombinant BAD0156 protein was characterized to confirm that this enzyme alone specifically hydrolyzes p-nitrophenyl-α-L-arabinofuranoside among the 24 p-nitrophenyl-glycosides examined. Among natural glycosides, α-1,5-linked arabino-oligosaccharides served as substrates, but arabinan, debranched arabinan, arabinoxylan, and arabinogalactan did not. A time course analysis of arabino-oligosaccharide hydrolysis indicated that BAD0156 is an exo-acting enzyme. These results suggest that BAD0156 is an α-L-arabinofuranosidase. This is the first report of a GH1 enzyme that acts specifically on arabinosides, providing information on GH1 substrate specificity.

Counterselection system for Geobacillus kaustophilus HTA426 through disruption of pyrF and pyrR.

Counterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system for Geobacillus kaustophilus HTA426, established through simultaneous disruption of the pyrF and pyrR genes. The pyrF gene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (ΔpyrF) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented with pyrF was prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, the pyrR gene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (ΔpyrF ΔpyrR) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented with pyrF was prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested that pyrF could serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous ß-galactosidase and α-amylase genes. The integrated genes were functionally expressed in G. kaustophilus and conferred new functions on the thermophile. This report describes the first establishment of a pyrF-based counterselection system in a Bacillus-related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression in G. kaustophilus. Our results also suggest a new strategy for establishment of counterselection systems.

Three inositol dehydrogenases involved in utilization and interconversion of inositol stereoisomers in a thermophile, Geobacillus kaustophilus HTA426.

Geobacillus kaustophilus HTA426, a thermophilic Bacillus-related species, utilizes some inositol stereoisomers, including myo-, d-chiro- and scyllo-inositols (MI, DCI and SI), as sole carbon sources. Within its genome are three paralogous genes that possibly encode inositol dehydrogenase. These genes are located in tandem within a large gene cluster containing an almost complete set of iol genes homologous to genes involved in inositol catabolism in Bacillus subtilis. Each of the three plausible inositol dehydrogenases was purified as a His(6)-tag fusion. The enzymes exhibited thermophilic activity, each with its own characteristic specificity for the inositol stereoisomers and cofactors. Northern blot and primer extension analyses revealed that the three enzymes were encoded by the same 5 kb polycistronic transcript and were induced simultaneously in the presence of MI. HTA426 was subjected to ethyl methanesulfonate (EMS) mutagenesis to isolate a mutant strain, PS8, which was not able to utilize MI. In PS8, inositol dehydrogenase activity was abolished along with the 5 kb transcript, suggesting that any of the three enzymes supports MI-dependent growth. Analysis of metabolites in HTA426 cells grown in the presence of MI revealed that substantial amounts of DCI and SI appeared intracellularly during the stationary phase, while only MI was present in PS8 cells, suggesting that interconversion of inositol stereoisomers may involve these three enzymes.