TNF receptor-associated factor 6 (TRAF6) plays a key role in the regulation of innate immune responses by mediating signals from both TNF receptors (TNFRs) and interleukin-1 receptors (IL-1Rs)/Toll-like receptors (TLRs). Here, we define a new role for TRAF6 in antagonizing cell death during TNF signaling. In TRAF6-deficient 3T3 (T6 À/À 3T3) cells, TNF stimulation leads to the accumulation of reactive oxygen species (ROS), which in turn results in prolonged c-Jun N-terminal kinase (JNK) activation and accelerated cell death. Furthermore, TNF-induced p65/RelA phosphorylation as well as transcriptional activity of nuclear factor-jB (NF-jB) was significantly downregulated in T6 À/À 3T3 cells. Interestingly, TRAF6 deficiency leads to constitutive phosphorylation and inactivation of glycogen synthase kinase 3b (GSK3b). Restoration of GSK3b activity through exogenous expression of a GSK3b constitutive active form rescued cell death in TRAF6-null 3T3 cells. These data suggest a role for TRAF6 in the maintenance of cell survival by regulating GSK3b activity in TNF signaling.
We read with interest the comments raised by Gong et al.1 on our recently published study, 2 and appreciate these important and critical remarks.We agree that contamination is a potential important problem with polymerase chain reaction (PCR)-based work. To avoid the possibility of contamination, we confirmed our data before completion of our manuscript. We performed the PCR experiment using new experimental reagents at a different laboratory. We also repeated the PCR to detect the fusion using two different set of primers, the new primer set producing longer amplicons than the old primer set. All these efforts produced identical results, confirming that our samples were not contaminated by PCR amplicons.As the direct fusion of one exon of CTLA4 to an exon of CD28 is less likely than fusion breakpoints within the introns, we confirmed direct fusion of the two exons at the genomic DNA level with other experiments before submitting our manuscript. As shown in Figure 1, direct fusion of exon 3 of CTLA4 to exon 4 of CD28 was confirmed by PCR using primers from intron 2 of CTLA4 and the 3' region of CD28 using genomic DNA from formalin-fixed, paraffin-embedded samples. PCR products were validated by Sanger sequencing. DNA from normal blood was used as a control as well as no template controls. These data confirm that the fusion is present at the DNA level and is not the result of contamination.We also tried to perform fluorescent in situ hybridization experiments. However it was difficult to design probes which can distinguish the fusion segment because the CD28 and CTLA4 genes are located very close to each other. As shown in our manuscript 2 in Online Supplementary Figure S5, the result was not helpful for delineating the fusion signal from the normal signal.
Introduction The aim of this study was to investigate the efficacy of changing sleep timing to afternoon-evening following nightshifts in hospital nurses with three rapid rotating shift schedules. Methods Hospital nurses with three rotating shift schedules were enrolled for a 1-month pre-intervention and a 1-month intervention study. During the Intervention, sleep timing following nightshifts was directed to afternoon-evening sleep for 8h time-in-bed (TIB) after 1 PM, and ad-lib sleep schedule for other shifts. Baseline and follow-up evaluation included sleep schedule, sleep duration, Epworth sleepiness scale (ESS), insomnia severity index (ISI) for each shift, Beck depression inventory (BDI), and Beck anxiety inventory (BAI). Sleep was assessed by sleep diary and actigraphy. Alertness during the night shift was evaluated using the Karolinska sleepiness scale (KSS) in the beginning and at the end of the shift by texts sent to their cell phones. The participants were asked to give feedback and a willingness to continue this intervention. Results A total of 26 subjects (30.7±8.5years, 25 female) finished the study among 29 nurses who participated in the study. The shift work was 6.5±8.0years. The mean morningness-eveningness scale was 42.1±8.0(31-62). TIB following nightshifts were 379.9±91.2 and 478.4±48.7 min for preintervention and intervention, respectively (p=0.001). Total sleep time (TST) was 328.0±91.0 vs. 361.0±70.4min, respectively following nightshifts (p=0.187, Cohen’s drm = 0.467). BDI, BAI, ESS, and ISI were significantly improved after the intervention. 60.7% and 49% of the participants reported improved alertness, and work efficiency during the nightshift. 17.9% and 42.9% of the participants reported increased sleep duration, and improved sleep quality after nightshift, respectively. Only eight participants were willing to continue the afternoon-evening sleep schedule following night shifts. KSS was not different between pre-intervention and intervention. Conclusion The afternoon-evening sleep schedule modestly increased total sleep time following nightshift. The overall mood, sleepiness and insomnia scale improved after the intervention although the alertness assessed by KSS failed to show the difference. The individual difference should be considered for applying afternoon-evening sleep for rapid rotating shift schedules. Support 2018 Research award grants from the Korean sleep research society and NRF-2019R1A2C1090643 funded by the Korean national research foundation
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