Original Article

99 cases were operated while we could not use antibiotics. The author traced X-ray photos on paper and measured areas of the peeled cavities with a planimeter. Results were as follows. 1) 66 cases had increasing stage and the rates were more than 30 %. 2) Cases with good developments showed larger original areas (50〜100cm^2) and smaller increasing rates (less than 30 %). 3) Also their X-ray photos showed coinciding or almost coinciding lines of the apices of lungs and the bases of cavities, but we had to take precautions against suppuration when they showed a horizontal line several days after operation. 4) Most of too high degree of adhesion or thickning of pleura did not show good results. When we found a cord which we must manage with some procedures by pneumolysis we must attend to suppuration too. 5)We ought to resect 4th or 5th rib more than 20 cm and 5th or 4th several cm supplementary. 6) As a method of constriction we commend the INVAGI.NATION method. 7) The author noticed in a considerable number of cases that the areas of cavities increased again after they kept long balanced stages

Atherosclerosis V, Proceeding of the Fifth International Symposium, A.M. Gotto, L.C. Smith, B. Allen, Spring Verlag, 1979(BOOK REVIEW)

Antiviral effect of micafungin on three strains of human rhinoviruses. H1HeLa cells were infected with human rhinovirus type 14 (A), 21 (B), or 71 (C) (100 CCID50) and immediately treated with indicated concentrations of micafungin. Three days after compound treatment antiviral activity was determined by the reduction of cytopathic effect using MTT assay. Cell viability of DMSO-treated cells was set to 0 % and that of uninfected cells was set to 100 %. (TIF 100 kb

sj-pdf-1-chc-10.1177_1367493520943772 – Supplemental Material for The predictors, barriers and facilitators to effective management of acute pain in children by emergency medical services: A systematic mixed studies review

Supplemental Material, sj-pdf-1-chc-10.1177_1367493520943772 for The predictors, barriers and facilitators to effective management of acute pain in children by emergency medical services: A systematic mixed studies review by Gregory A Whitley, Pippa Hemingway, Graham R Law, Arwel W Jones, Ffion Curtis and Aloysius N Siriwardena in Journal of Child Health Care</p

Identification of a Novel Function of CX-4945 as a Splicing Regulator

<div><p>Alternative splicing is a nearly ubiquitous versatile process that controls gene expression and creates numerous protein isoforms with different functions from a single gene. The significance of alternative splicing has been confirmed by the increasing number of human diseases that are caused by misregulation of splicing events. Very few compounds, however, have been reported to act as inhibitors of alternative splicing, and their potential clinical use needs to be evaluated. Here, we report that CX-4945, a previously well-characterized inhibitor of casein kinase 2 (CK2) and a molecule currently in clinical trials (Phase II) for cancer treatment, regulates splicing in mammalian cells in a CK2-independent manner. Transcriptome-wide analysis using exon array also showed a widespread alteration in alternative splicing of numerous genes. We found that CX-4945 potently inhibits the Cdc2-like kinases (Clks) <i>in vitro</i> and in turn, leads to suppression of the phosphorylation of serine/arginine-rich (SR) proteins in mammalian cells. Surprisingly, the overall efficacy of CX-4945 on Clks (IC₅₀ = 3–90 nM) was stronger than that of TG-003, the strongest inhibitor reported to date. Of the Clks, Clk2 was most strongly inhibited by CX-4945 in an ATP-competitive manner. Our research revealed an unexpected activity of the drug candidate CX-4945 as a potent splicing modulator and also suggested a potential application for therapy of diseases caused by abnormal splicing.</p></div

The widespread effect of CX-4945 on alternative splicing of numerous genes.

<p>(A) 293T cells were treated with DMSO or 10 µM CX-4945 for 12 hours, and total RNA was prepared. The cDNA was synthesized and further processed for exon array as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094978#s2" target="_blank">Materials and Methods</a>. All experiments were performed three times, and representative heat maps of selected transcripts from the exon arrays are shown. Selected exons for each mRNA are highlighted with black underlines. (B) RT-PCR analysis was performed for the ELL2, SPAST, QRSL1, PRPSAP2, CPEB1, USP38, TRIP4, CTDSPL2, WDFY1 and ZCCHC2 genes with the same RNA used for the exon arrays. The exonic structure of the RT-PCR products and the exonic location of PCR primers are indicated with tandem rectangles and arrowheads, respectively. PCR products were quantified using ImageJ software, and the ratios of skipped product (skipped/total PCR product) were calculated. The average and SD values were determined from two independent experiments and are presented below the PCR data.</p

CX-4945 modulates SR protein phosphorylation via direct inhibition of Clks.

<p>(A) Total protein extracts from 293T cells treated with CX-4945 (1 and 10 µM) or TG-003 (10 and 50 µM) for 1 hour were separated by SDS-PAGE, and phosphorylated SR proteins were monitored by western blotting using the phosphoSR monoclonal antibody (1H4). SRSF1, SRSF4, SRSF9, and hnRNP A1 proteins were also monitored as controls. Western blotting was performed twice, and representative data are presented. (B) The phosphorylated SR proteins in (A) were quantified, and amounts of each protein relative to those of DMSO-treated samples are shown. The average and SD values were determined from two independent experiments. (C) The potent inhibition of Clks by CX-4945 was observed in <i>in vitro</i> kinase assays conducted by Life Technologies using recombinant human Clk1, Clk2, Clk3, SRPK1, SRPK2, and Ck2 α proteins. The average and SD values were determined from two independent assays. The IC₅₀ values were determined using PRISM software. Detailed experimental procedures are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094978#s2" target="_blank">Materials and Methods</a>.</p

CX-4945 affects alternative splicing in a CK2-independent manner.

<p>(A) Two other inhibitors of CK2, TBB and TBCA, were also tested to determine whether splicing regulation by CX-4945 is related to CK2 activity. 293T cells were treated with 10 µM CX-4945 or 100 µM TBB or and TBCA for 12 hours. Inhibitory activities of CX-4945, TBB, and TBCA on CK2 were assessed by examination of phosphorylation of AKT(S129), the major substrate of CK2. Total levels of AKT were also monitored as a control. hnRNP A1 was used as a loading control. (B) Alternatively spliced CK2 α′, ELL2, CPEB1, PRPSAP2, and QRSL1 mRNAs were examined by RT-PCR. PCR products of normal and alternatively spliced isoforms are denoted by black arrowheads. An extra PCR product of the QRSL1 gene appeared in TBB-treated samples and is denoted by an asterisk. All experiments were performed twice, and representative data for each are presented.</p

Effect of CX-4945 on pre-mRNA splicing.

<p>(A) CX-4945 induces expression of an unexpected CK2 α′ transcript, CK2 α′△. RT-PCR corresponding to exons 5–6–7 was performed with total RNA from DMSO- or CX-4945-treated 293T cells. (B) CX-4945 rapidly induces the expression of CK2 α′△. 293T cells were treated with CX-4945 for the times indicated prior to RNA preparation and RT-PCR analysis. GAPDH mRNA was analyzed as a control. (C) Analysis of the DNA sequence of CK2 α′△ revealed that it represents the exon6-skipped product (CK2 α′△exon6) of CK2 α′ pre-mRNA. The normally spliced (CK2 α′) and alternatively spliced (CK2 α′△exon6) products are illustrated schematically.</p

ATP-competitive inhibition of Clks by CX-4945.

<p>(A) Diagram of the chemical structure of CX-4945 (B) Competition of CX-4945 with ATP for inhibition of Clk2 (C) Structural model of the Clk2 in complex with CX-4945. CX-4945 was predicted to bind to the ATP-binding site of Clk2. (D) The predicted binding mode between CX-4945 and Clk2. Amino acids in the active site are denoted by yellow sticks, and CX-4945 is indicated by green sticks. Hydrogen bonds are denoted as dotted black lines, and oxygen and nitrogen atoms are indicated as red and blue sticks, respectively.</p

Additional file 2: Figure S2. of Antiviral activity of micafungin against enterovirus 71

Micafungin inhibits the replication of CVB replicon. (A) Vero cells were transfected with in vitro transcribed CVB3 replicon RNAs, instantly treated with indicated concentrations of micafungin for 8 hours and then assayed for firefly luciferase activity. Luciferase activity of DMSO-treated cells was set to 100 %. (B) At the same condition another set of CVB3 replicon-transfected cells were assayed for cell viability by using CellTiter-Glo reagent. Activity of DMSO-treated cells was set to 100 %. (TIF 100 kb