Ewing sarcoma is the second most common pediatric bone cancer, with a 5-year survival rate for metastatic disease of only 20%. Recent work indicates that survival is strongly correlated with high levels of tumor-infiltrating lymphocytes (TIL), whose abundance is associated with IFN-inducible chemokines CXCL10 and CCL5. However, the tumor-intrinsic factors that drive chemokine production and TIL recruitment have not been fully elucidated. We previously showed that ubiquitin-specific protease 6 (USP6) directly deubiquitinates and stabilizes Jak1, thereby inducing an IFN signature in Ewing sarcoma cells. Here, we show that this gene set comprises chemokines associated with immunostimulatory, antitumorigenic functions, including CXCL10 and CCL5. USP6 synergistically enhanced chemokine production in response to exogenous IFN by inducing surface upregulation of IFNAR1 and IFNGR1. USP6-expressing Ewing sarcoma cells stimulated migration of primary human monocytes and T lymphocytes and triggered activation of natural killer (NK) cells in vitro. USP6 inhibited Ewing sarcoma xenograft growth in nude but not NSG mice and was accompanied by increased intratumoral chemokine production and infiltration and activation of NK cells, dendritic cells, and macrophages, consistent with a requirement for innate immune cells in mediating the antitumorigenic effects of USP6. High USP6 expression in patients with Ewing sarcoma was associated with chemokine production, immune infiltration, and improved survival. This work reveals a previously unrecognized tumor-suppressive function for USP6, which engenders an immunostimulatory microenvironment through pleiotropic effects on multiple immune lineages. This further raises the possibility that USP6 activity may be harnessed to create a "hot" tumor microenvironment in immunotherapy.Significance: This study reveals a novel tumor-suppressive function for USP6 by inducing an immunostimulatory microenvironment, suggesting that USP6 activity may be exploited to enhance immunotherapy regimens.

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Most, but not all, homologous genetic recombination in bacteria is mediated by the RecA recombinase. The mechanistic origin of RecA‐independent recombination has remained enigmatic. Here, we demonstrate that the RarA protein makes a major enzymatic contribution to RecA‐independent recombination. In particular, RarA makes substantial contributions to intermolecular recombination and to recombination events involving relatively short (<200 bp) homologous sequences, where RecA‐mediated recombination is inefficient. The effects are seen here in plasmid‐based recombination assays and in vivo cloning processes. Vestigial levels of recombination remain even when both RecA and RarA are absent. Additional pathways for RecA‐independent recombination, possibly mediated by helicases, are suppressed by exonucleases ExoI and RecJ. Translesion DNA polymerases may also contribute. Our results provide additional substance to a previous report of a functional overlap between RecA and RarA.

Objective:The objective of this study was to chemically evaluate precipitate formation on irrigation by different concentrations of chlorhexidine (CHX) and alexidine (ALX) with sodium hypochlorite (NaOCl).Materials and Methods:Six test tubes were prepared with 1 ml of 4% NaOCl. One milliliter of 2%, 1%, 0.5%, and 0.25% ALX was added to the first four, and in the last two, 1 ml of 2% CHX and 0.2% CHX was added, respectively. Samples were observed for color changes or precipitates at multiple time intervals. All solutions were then centrifuged at 1000 rpm for 10 min and re-examined for precipitates. This process was repeated twice. Fifty freshly extracted premolars were biomechanically prepared, dried, divided into two groups, and irrigated with 10 ml of 4% NaOCl and 10 ml of 2% ALX (Group 1) and 10 ml of 4% NaOCl and 10 ml of 2% CHX (Group 2). These samples were sectioned and observed for precipitates on the dentinal surfaces by scanning electron microscopy (SEM).Results:The color of the solution of ALX and NaOCl stayed transparent and no precipitate was observed. A color change was noted immediately on mixing CHX and NaOCl which did not change with time. Precipitates were only observed in the solutions of CHX with NaOCl and after centrifuging them. SEM views also showed dense precipitates covering the dentinal surface and occluding the dentinal tubules in Group 2.Conclusion:The interaction of ALX and NaOCl does not produce precipitates which together with its better antimicrobial action make ALX a more effective and safer replacement for CHX as an adjunctive endodontic irrigant.