Establishment of the improved colonization of Escherichia coli laboratory strain in the intestine mediated by single gene deletion.

In light of the emerging importance of the gut microbiome in human health, there is a need to improve the colonization efficiency of therapeutic bacteria called probiotics. Despite their recognized potential, artificially administered bacteria exhibit poor colonization in the intestine, limiting their therapeutic efficacy. Addressing this challenge requires innovative strategies; however, reported examples are limited. In nature, including in the intestinal tract, bacteria live via biofilm formation. Recently, it has been reported that RNase I, a member of the RNase T2 family conserved among almost all species, including bacteria, inhibits biofilm formation in Escherichia coli. In this study, we focus on these results and investigate the relationship between high biofilm formation and intestinal attachment using a non-settling E. coli laboratory strain as a probiotic model. The intestinal colonization abilities were evaluated through a microfluidic device mimicking the intestinal tract and through oral administration to mice. The in vitro and in vivo experiments showed that the E. coli strain lacking RNase I exhibited remarkable stability in intestinal colonization. We investigated the observation of colonization using fluorescence in situ hybridization, and inoculated E. coli cells were aggregated with the gut microbiome in the cecum and colon. This study proposes a technique to improve the intestinal colonization of bacteria by simply manipulating a single gene disruption, and it is expected to contribute to future research on the colonization of useful bacteria.

Five multicopy gene family genes expressed during the maternal-to-zygotic transition are not essential for mouse development.

Upon fertilization, oocytes transform into totipotent and pluripotent cleavage stage cells through the maternal-to-zygotic transition (MZT), which is regulated by maternal factors and zygotic genome activation (ZGA). Here, we investigated the in vivo function of 16 genes expressed with strong biases in oocytes and cleavage stage embryos by generating knockout (KO) mice. These MZT-associated genes are conserved across many mammalian species and include five multicopy gene family genes: the Nlrp9, Khdc1, Rfpl4, Trim43, and Zscan5 genes. Intercrosses between female KO and male KO mice, including Nlrp9a/b/c triple KO (TKO), Khdc1a/b/c TKO, Rfpl4a/b double KO (DKO), Trim43a/b/c TKO, and Zscan5b KO mice led to the birth to healthy offspring that in turn produced healthy offspring. Our study not only demonstrated that these MZT-associated genes are not essential for mouse development, but also provides valuable resources for analyzing the functions of these genes in other genetic backgrounds, in the presence of stressors, and under pathogenic conditions.

Involvement of Nlrp9a/b/c in mouse preimplantation development.

Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing proteins (NRLPs) are central components of the inflammasome. Accumulating evidence has shown that a reproductive clade of NRLPs is predominantly expressed in oocyte to cleavage stage embryos and participates in mammalian preimplantation development as a component of a multiprotein complex known as the subcortical maternal complex (SCMC). Nlrp9s belong to the reproductive class of NLRPs; Nlrp9b is unique in acting as an inflammasome against rotavirus in intestines. Here we generated mice carrying mutations in all three members of the Nlrp9a/b/c gene (Nlrp9 triple mutant (TMut) mice). When crossed with WT males, the Nlrp9 TMut females were fertile, but deliveries with fewer pups were increased in the mutants. Consistent with this, blastocyst development was retarded and lethality to the preimplantation embryos increased in the Nlrp9 TMut females in vivo. Under in vitro culture conditions, the fertilized eggs from the Nlrp9 TMut females exhibited developmental arrest at the two-cell stage, accompanied by asymmetric cell division. By contrast, double-mutant (DMut) oocytes (any genetic combination) did not exhibit the two-cell block in vitro, showing the functional redundancy of Nlrp9a/b/c. Finally, Nlrp9 could bind to components of the SCMC. These results show that Nlrp9 functions as an immune or reproductive NLRP in a cell-type-dependent manner.

High susceptibility of atherosclerotic coronary arteries to the onset of vasospasm and angina pectoris-like symptoms due to coronary spasm in WHHLMI rabbits.

We examined the relationship between atherosclerosis and the provocation of coronary spasm as well as the influence of coronary spasm on the onset of acute ischemic myocardial disease. Coronary spasm was provoked in anesthetized normal Japanese white (JW) rabbits and myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for coronary atherosclerosis and myocardial infarction, by injecting ergonovine during the infusion of norepinephrine through a marginal ear vein. A decrease in contrast flow in the left circumflex artery was observed on coronary angiograms. Ischemic changes were observed on the electrocardiograms of 29% (2/7) of JW and 79% (27/34, P=0.007) of WHHLMI rabbits. The frequency of coronary spasm was significantly high in rabbits with severe coronary plaques showing diffuse lesions. Left ventricle motility in vasospasm-positive rabbits, which was evaluated with echocardiograms, was decreased by 29% following the ergonovine injection (P<0.001), and every serum ischemic marker markedly increased 4 h after the provocation of vasospasm. These results demonstrate that atherosclerotic coronary arteries are positively related to the provocation of vasospasm, and vasospasm in severe atherosclerotic coronary segments evokes angina pectoris-like findings and/or non-fatal myocardial infarction. WHHLMI rabbits may be a novel animal model for angina pectoris and acute ischemic heart disease.