Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases.

Removal of ribonucleotides (rNMPs) incorporated into the genome by the ribonucleotide excision repair (RER) is essential to avoid genetic instability. In eukaryotes, the RNaseH2 is the only known enzyme able to incise 5' of the rNMP, starting the RER process, which is subsequently carried out by replicative DNA polymerases (Pols) δ or ϵ, together with Flap endonuclease 1 (Fen-1) and DNA ligase 1. Here, we show that the DEAD-box RNA helicase DDX3X has RNaseH2-like activity and can support fully reconstituted in vitro RER reactions, not only with Pol δ but also with the repair Pols ß and λ. Silencing of DDX3X causes accumulation of rNMPs in the cellular genome. These results support the existence of alternative RER pathways conferring high flexibility to human cells in responding to the threat posed by rNMPs incorporation.

Squaramide-Naphthalimide Conjugates as "Turn-On" Fluorescent Sensors for Bromide Through an Aggregation-Disaggregation Approach.

The syntheses of two new squaramide-naphthalimide conjugates (SQ1 and SQ2) are reported where both compounds have been shown to act as selective fluorescence "turn on" probes for bromide in aqueous DMSO solution through a disaggregation induced response. SQ1 and SQ2 displayed a large degree of self-aggregation in aqueous solution that is disrupted at increased temperature as studied by 1H NMR and Scanning Electron Microscopy (SEM). Moreover, the fluorescence behavior of both receptors was shown to be highly dependent upon the aggregation state and increasing temperature gave rise to a significant increase in fluorescence intensity. Moreover, this disaggregation induced emission (DIE) response was exploited for the selective recognition of certain halides, where the receptors gave rise to distinct responses related to the interaction of the various halide anions with the receptors. Addition of F- rendered both compounds non-emissive; thought to be due to a deprotonation event while, surprisingly, Br- resulted in a dramatic 500-600% fluorescence enhancement thought to be due to a disruption of compound aggregation and allowing the monomeric receptors to dominate in solution. Furthermore, optical sensing parameters such as limits of detection and binding constant of probes were also measured toward the various halides (F-, Cl-, Br-, and I-) where both SQ1 and SQ2 were found to sense halides with adequate sensitivity to measure µM levels of halide contamination. Finally, initial studies in a human cell line were also conducted where it was observed that both compounds are capable of being taken up by HeLa cells, exhibiting intracellular fluorescence as measured by both confocal microscopy and flow cytometry. Finally, using flow cytometry we were also able to show that cells treated with NaBr exhibited a demonstrable spectroscopic response when treated with either SQ1 or SQ2.

Hepatitis C virus (HCV)-induced suppressor of cytokine signaling (SOCS) 3 regulates proinflammatory TNF-α responses.

TNF-α is a proinflammatory cytokine, dramatically elevated during pathogenic infection and often responsible for inflammation-induced disease pathology. SOCS proteins are inhibitors of cytokine signaling and regulators of inflammation. In this study, we found that both SOCS1 and SOCS3 were transiently induced by TNF-α and negatively regulate its NF-κB-mediated signal transduction. We discovered that PBMCs from HCV-infected patients have elevated endogenous SOCS3 expression but less TNF-α-mediated IκB degradation and proinflammatory cytokine production than healthy controls. HCV protein expression in Huh7 hepatocytes also induced SOCS3 and directly inhibited TNF-α-mediated IL-8 production. Furthermore, we found that SOCS3 associates with TRAF2 and inhibits TRAF2-mediated NF-κB promoter activity, suggesting a mechanism by which SOCS3 inhibits TNF-α-mediated signaling. These results demonstrate a role for SOCS3 in regulating proinflammatory TNF-α signal transduction and reveal a novel immune-modulatory mechanism by which HCV suppresses inflammatory responses in primary immune cells and hepatocytes, perhaps explaining mild pathology often associated with acute HCV infection.