Tabernacles of the Spirit

In the classic tradition of the exploratory essay, George Gammack examines the theme of community in this paper. He details varied aspects of the creation of community among those who are retired, taking as its focus the Men’s Sheds movement. The paper explores the relationship between persons and community in later years, looking in particular at how those with a lifetime’s worth of skills and knowledge can continue to contribute to the life of a community. Along the way we are introduced to the work of authors such as Charles Taylor, Richard Niebuhr, Primo Levi, Seamus Heaney and Richard Sennett on the subject of work and what comes after it.Publisher PD

Multivariable analyses for associations between PR and the risk of RA, SLE, or SS among subgroups according to age, sex, and CCI.

<p>Multivariable analyses for associations between PR and the risk of RA, SLE, or SS among subgroups according to age, sex, and CCI.</p

Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways

<div><p>Avian reovirus (ARV) protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128) of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication.</p></div

A rescue experiment by overexpression of EGFP-Tpr in ARV-infected and p17-transfected vero cells as well as examination of AKT required for p21 phosphorylation.

<p>(A) To carry out a rescue experiment by overexpression of EGFP-Tpr in ARV-infected and p17-transfected vero cells, cells were transfected with the EGFP-Tpr plasmids for 48 hours, and then either infected with ARV at an MOI of 10 or p17 transfection for 24 hours. Whole cell lysates were collected for Western blot assays. Histone H2A was used as a loading control. The activation and inactivation folds indicated below each lane were normalized against those in mock controls (cell alone). The levels of indicated proteins in the mock control were considered 1-fold. (B) Representative images of vero cells transfected with plasmids overexpressing EGFP-Tpr. Vero cells were transfected with negative controls (mock and vector only) or EGFP-Tpr plasmid for 48 hours and then visualized by immunofluorescence following nuclear DAPI staining. In addition, another set of cells were transfected with EGFP-Tpr for 48 hours and then transfected with p17 for 24 hours. Cells were fixed and processed for immunofluorescence staining of p17. Colocalization of EGFP-Tpr and p17 was observed under a fluorescence microscope. Scale bar: 10 <i>um</i>. (C) To examine whether AKT kinase activity is directly required for p21, we used an Akt DN to inhibit AKT in Vero cells. The levels of p-p21 (T145) in the nucleus were examined in ARV-infected and p17-transfected cells in the presence or absence of an Akt DN. Histone H2A was used as a loading control.</p

p17 negatively regulates ERK and cyclin D1 and positively regulates Rb through Tpr/p53/PTEN and Tpr/p53/p21 pathways.

<p>(A-B)Vero cells were transfected with pcDNA3.1-p17 or pcDNA3.1 (vector only) for 24 hours. The results of Western blot analysis of cellular fractions from the cytoplasm and nucleus (A) or whole cell lysates (B) at the indicated time points are shown. Phosphorylation and protein levels were determined by immunoblotting with the indicated antibodies. (C) To study whether the p17 mutant (1–118) which does not possess a NLS can affect the levels of p-ERK, cyclin D1, and CDK4 in the nucleus, vero cells were transfected with the negative control p17 mutant (1–118) for 24 hours. (D) To confirm whether PTEN is the upstream signaling that regulates the above molecules, shRNA-mediated blockade of PTEN was performed. Vero and DF-1 cells were co-transfected with pcDNA3.1-p17 and PTEN shRNAs for 24 hours followed by Western blot analysis with indicated antibodies. In the negative controls, cells were also co-transfected with p17 and respective negative controls (pGFP-V-RS and Scramble shRNA plasmids) for 24 hours. Phosphorylation and protein levels were determined by immunoblotting with the indicated antibodies. The protein levels were normalized to that for β-actin (panels A, B, C) or Histone H2A (panel A). The activation and inactivation folds indicated below each lane were normalized against those at 0 h or in mock control. The levels of indicated proteins at 0 h or in mock control (cell only) were considered 1-fold. The representative data are from three independent experiments.</p

Primers used in this study for amplification of the respective targeted genes.

<p>F: forward; R: reverse</p><p>* Uderlines indicate the restriction sites designed in the indicated primers</p><p>Primers used in this study for amplification of the respective targeted genes.</p

p17 functions as a Tpr suppressor leading to p53 and p21 nuclear accumulation.

<p>(A) Both Tpr and p53 levels in mock control (Vero cell only), pcDNA3.1 (mock transfection)-, pcDNA3.1-p17-transfected, and ARV-infected cells were examined. Whole cell lysates and nuclear extracts were collected at the indicated time points for Western blot assays. (B) To examine whether the Tpr transcription was down-regulated by p17, the Tpr mRNA levels in ARV-infected and pcDNA3.1-p17-transfected Vero cells were compared with those in mock treated cells. In semi-quantitative RT-PCR amplification of the p17 and Tpr genes, Vero cells were transfected with pcDNA3.1-p17 or infected with ARV at an MOI of 10. The pcCNA3.1-p17 transfected or ARV-infected cells were collected at 24 hours postinfection (hpi), and total RNAs were extracted for semi-quantitative RT-PCR. After electrophoretic separation in an agarose gel, PCR products were stained with ethidium bromide. Mock transfection (vector only) and mock infection (cell alone) were included as negative controls. Graph shown represents the mean± SD calculated from three independent experiments. (C) To study whether the p17 mutant (1–118), which does not possess a NLS, can influence the levels of p-p53 and p-p21 in the nucleus, vero cells were transfected with the p17 mutant (1–118) plasmid (negative control) for 24 hours. (D) To study whether Tpr depletion affects p53, p21, and PTEN nuclear accumulation, nuclear extracts from ARV-infected and pcDNA3.1-p17-transfected Vero cells were collected for Western blot assays. Vero cells were transfected with Tpr shRNA for 6 hours before being infected with ARV at an MOI of 10 for 18 hours. In a parallel experiment, Vero cells were co-transfected with pcDNA3.1-p17 and Tpr shRNA plasmid for 18 hours. Nuclear extracts were collected for Western blot assays using the indicated antibodies. Either actin or histone H2A was used as loading controls. The activation and inactivation folds indicated below each lane were normalized against those at 0 h (panel A) or in mock controls (cell alone) (panels C and D). The levels of indicated proteins at 0 h or in the mock controls (cell alone) were considered 1-fold.</p

Risk of autoimmune rheumatic diseases in patients with palindromic rheumatism: A nationwide, population-based, cohort study - Fig 1

The cumulative incidences of RA (A), SLE (B), and SS (C) in the PR group and the non-PR group.</p

p17 negatively regulates PI3K/AKT/mTOR signaling pathway.

<p>(A-B) Vero cells were transfected with pcDNA3.1-p17 and pcDNA3.1 (vector only) plasmid, respectively for 24 hours. Whole cell lysates were collected at the indicated time points, and the levels of PI3K and its downstream molecules were examined by Western blot assay with the indicated antibodies. (B)Vero and DF-1 cells were co-transfected with both pcDNA3.1-p17 and p53 shRNAs for 24 hours, followed by Western blot analysis with indicated antibodies. Cells were also co-transfected with pCDNA3.1- p17 and respective negative controls (scrambled shRNAs and pGFP-V-RS vector) for 24 hours. (C) To study whether the negative control p17 mutant (1–118) can affect the levels of p-PTEN, p-AKT, p-mTOR, and LC3-II, vero cells were transfected with the p17 mutant (1–118) plasmid for 24 hours. Similar results were obtained from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins at 0 h or mock were considered 1-fold.</p

Knockdown of Tpr is beneficial for virus replication.

<p>(A)To determine the effect of Tpr on ARV replication, depletion of Tpr with shRNA was carried out in ARV-infected Vero cells. The downstream molecules (p53, PTEN and LC3) of Tpr were also depleted using the respective shRNAs. All data shown represent the mean± SD calculated from three independent experiments. (B) In the present study, the effects of PI3K inhibitor (LY294002, 10 uM), mTORC1 inhibitor (rapamycin; 5 uM), and inhibitor of autophagy (3MA) on ARV replication were examined. Data shown represent the mean± SD calculated from three independent experiments. (C) In the case of CDK 4, vero cells were infected with ARV at a MOI of 5 for 3 hours, followed by CDK 4 shRNA transfection for 18 hours. All data shown represent the mean± SD calculated from three independent experiments.</p