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During injury, monocytes are recruited from the circulation to inflamed tissues and differentiate locally into mature macrophages, with prior reports showing that cavity macrophages of the peritoneum and pericardium invade deeply into the respective organs to promote repair. Here we report a dual recombinase-mediated genetic system designed to trace cavity macrophages in vivo by intersectional detection of two characteristic markers. Lineage tracing with this method shows accumulation of cavity macrophages during lung and liver injury on the surface of visceral organs without penetration into the parenchyma. Additional data suggest that these peritoneal or pleural cavity macrophages do not contribute to tissue repair and regeneration. Our in vivo genetic targeting approach thus provides a reliable method to identify and characterize cavity macrophages during their development and in tissue repair and regeneration, and distinguishes these cells from other lineages.

Background To explore the optimum fermentation conditions for tobacco leaves and also screen the microbiota and metabolites that are beneficial for fermentation. Methods Tobacco leaves were fermented at 25 °C, 35 °C, and 45 °C for 2, 4, and 6 weeks, respectively. For identification of the best fermentation temperature, physicochemical properties and sensory quality of fermented tobacco were investigated. Subsequently, based on the appropriate temperature, 16 s rRNA sequencing and metabolomics analysis of tobacco were performed to monitor the change of microbes and metabolites during fermentation process (from 2 to 6 weeks). Results Sensory quality analysis indicated that fermentation at 45 °C for 6 weeks represented the optimum condition. Metabolomics analysis showed that a total of 415 metabolites were annotated. The increase of fermentation period led to significant changes of metabolites. Results revealed an increase in concentration of L-phenylalanine and sphingosine as well as decreased concentration of betaine and phytosphingosine with the prolongation of fermentation period (2 to 6 weeks). Distinct changes in the microbiota were also observed with prolongation of the fermentation time. Results revealed that Pseudomonas, Pantoea, and Burkholderia were dominant bacteria in fermentation at 45 °C for 6 weeks. With the extension of the fermentation time, the abundance of Pseudomonas increased, while that of Sphingomonas and Methylobacterium decreased. Furthermore, microbiota profiles were tightly relevant to the altered metabolites, especially compounds involved in the sphingolipid metabolism. Conclusion Suitable fermentation conditions were 45 °C for 6 weeks; phytosphingosine and sphingosine might affect tobacco fermentation via the sphingolipid metabolism pathway. This study provides a theoretical basis for guiding tobacco fermentation and gives insights into reducing harmful substances during tobacco fermentation.

Prolyl hydroxylase 3 (PHD3) has initially been reported to hydroxylase hypoxia-inducible factor α (HIFα) and mediate HIFα degradation. More recent studies have shown that, in addition to HIFα, PHD3 has also other substrates. Moreover, pHD3 is believed to act as a tumor suppressor, but the underlying mechanism remains to be elucidated. Here, we demonstrate that PHD3 stabilizes p53 in a hydroxylase-independent manner. We found that PHD3 overexpression increases and PHD3 knockdown decreases p53 levels. Mechanistically, PHD3 bound MDM2 proto-oncogene (MDM2) and prevented MDM2 from interacting with p53, thereby inhibiting MDM2-mediated p53 degradation. Interestingly, we found that PHD3 overexpression could enhance p53 in the presence of the prolyl hydroxylase inhibitor dimethyloxalylglycine, and the prolyl hydroxylase activity-deficient variant PHD3-H196A also inhibited the p53-MDM2 interaction and stabilized p53. Genetic ablation of PHD3 decreased p53 protein levels in mice intestinal epithelial cells, but a genetic knockin of PHD3-H196A did not affect p53 protein levels in vivo. These results suggest that the prolyl hydroxylase activity of PHD3 is dispensable for its ability to stabilize p53. We found that both PHD3 and PHD3-H196A suppress the expression of the stem cell-associated gene NANOG and inhibited the properties of colon cancer stem cells through p53. Our results reveal an additional critical mechanism underlying the regulation of p53 expression and highlight that PHD3 plays a role in the suppression of colon cancer cell stemness in a hydroxylase-independent manner.

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