Macrophages (Mφ) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of Mφ to trigger transient activation of monocytes in peritumoral stroma. We showed that a fraction of monocytes/Mφ in peritumoral stroma, but not in cancer nests, expresses surface PD-L1 (also termed B7-H1) molecules in tumors from patients with hepatocellular carcinoma (HCC). Monocytes activated by tumors strongly express PD-L1 proteins with kinetics similar to their activation status, and significant correlations were found between the levels of PD-L1+ and HLA-DRhigh on tumor-infiltrating monocytes. Autocrine tumor necrosis factor α and interleukin 10 released from activated monocytes stimulated monocyte expression of PD-L1. The PD-L1+ monocytes effectively suppressed tumor-specific T cell immunity and contributed to the growth of human tumors in vivo; the effect could be reversed by blocking PD-L1 on those monocytes. Moreover, we found that PD-L1 expression on tumor-infiltrating monocytes increased with disease progression, and the intensity of the protein was associated with high mortality and reduced survival in the HCC patients. Thus, expression of PD-L1 on activated monocytes/Mφ may represent a novel mechanism that links the proinflammatory response to immune tolerance in the tumor milieu.

The full scale of human miRNome in specific cell or tissue, especially in cancers, remains to be determined. An in-depth analysis of miRNomes in human normal liver, hepatitis liver, and hepatocellular carcinoma (HCC) was carried out in this study. We found nine miRNAs accounted for ∼88.2% of the miRNome in human liver. The third most highly expressed miR-199a/b-3p is consistently decreased in HCC, and its decrement significantly correlates with poor survival of HCC patients. Moreover, miR-199a/b-3p can target tumor-promoting PAK4 to suppress HCC growth through inhibiting PAK4/Raf/MEK/ERK pathway both in vitro and in vivo. Our study provides miRNomes of human liver and HCC and contributes to better understanding of the important deregulated miRNAs in HCC and liver diseases.

Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme composed of two separately purifiable component proteins encoded by the structural genes nifH, nifD, and nifK. Deletion of the Azotobacter vinelandii nifS gene lowers the activities of both nitrogenase component proteins. Because both nitrogenase component proteins have metallocluster prosthetic groups that are composed of iron-and sulfur-containing cores, this result indicated that the nifS gene product could be involved in the mobilization of the iron or sulfur required for metallocluster formation. In the present work, it is shown that NIFS is a pyridoxal phosphatecontaining homodimer that catalyzes the formation ofL-alanine and elemental sulfur by using L-cysteine as substrate. NIFS activity is extremely sensitive to thiol-specific alkylating reagents, which indicates the participation of a cysteinyl thiolate at the active site. Based on these results we propose that an enzyme-bound cysteinyl persulfide that requires the release of the sulfur from the substrate L-cysteine for its formation ultimately provides the inorganic sulfide required for nitrogenase metaflocluster formation. The recent discovery of nifSlike genes in non-nitrogen-fiing organisms also raises the possibility that the reaction catalyzed by NIIFS represents a universal mechanism that involves pyridoxal phosphate chemistry, in the mobilization of the sulfur required for metailocluster formation.The reduction in both A. vinelandii nitrogenase component protein activities as a result of nijS deletion, which has also been reported for Klebsiella pneumoniae (6), could not be attributed to a regulatory effect (3). Thus, because both Fe protein and MoFe protein activities were affected by deletion of nijf , and the common feature of both component proteins is that they contain metalloclusters, we have considered that NIFS might be involved in the acquisition or mobilization of the inorganic Fe or sulfur required for metallocluster formation. This possibility is supported by the observation that cell pellets of diazotrophically grown A. vinelandii nifS mutants are pale tan rather than the characteristic dark brown of wild type. The dark color of diazotrophically grown wild-type A. vinelandii is attributed to the metalloclusters contained within the nitrogenase component proteins that form a substantial portion of the soluble protein fraction.As a strategy to elucidate the function of the NIFS polypeptide, we chose to produce large amounts of it in Escherichia coli to facilitate its purification and biochemical characterization. In the present study, we have purified the NIFS protein, demonstrated that a specific reaction is catalyzed by NIFS, and proposed a biochemical function for NIFS activity in relation to metallocluster formation. In addition, we discuss the possibility that the reaction catalyzed by NIFS represents a universal mechanism for the mobilization of the sulfur required for metallocluster formation. Many proteins that have important electron tr...

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