Corporate governance : external auditors and their opinions.

The increasing awareness and emphasis on corporate governance may be traced to the occurrence of many company failures over the past few years. The term corporate governance can be defined as the way corporate entities are governed, and the issues facing the board of directors, such as the interaction with top management, and relationships with the owners and others interested in the affairs of the company, including creditors, debt financiers, analysts, auditors and corporate regulators. Our study was conducted to identify the opinions of external auditors on various issues of corporate governance

Ethyl pyruvate inhibits HMGB1 release from DV-infected cells in dose-dependent manner.

<p>A, cell viability assay of K562 or PBM cells treated with 1, 3 and 5 mM of EP for 3 days. The percentages of viable K562 and PBM cells were calculated in relation to its untreated control groups. B, immunofluorescence analysis of EP-treated DV-infected K562 and PBM cells to visualize the sub-cellular localization of HMGB1 (stained green) in these cells. DV antigens were stained red and the cell nuclei were stained blue. C, K562 and PBM cells were infected with DV (M.O.I. of 1) and the cells were treated with EP at 0 hr p.i. Cell culture supernatants of DV-infected K562 and PBM cells were harvested 3 d.p.i. and concentrated for the detection of HMGB1 by Western blotting. The fold intensity of DV-infected K562 and PBM cells without EP treatment were assigned with a value of 1. The relative fold difference in the HMGB1 intensities of EP-treated DV-infected samples, mock-infected and LPS-treated cells were compared in relation to that of the DV-infected K562 or PBM cells. D, amount of HMGB1 in the cell culture supernantants was quantified using ELISA. * denotes p-value <0.05 for T-tests comparing the mean amount of HMGB1 detected in K562 cell culture supernatants to that of the DV-infected K562 cells. ⧫ denotes p-value <0.05 for T-tests comparing the mean amount of HMGB1 detected in PBM cell culture supernatants to that of the DV-infected PBM cells. E, plaque assays were conducted on the cell culture supernatants of DV-infected EP-treated K562 cells at 3 d.p.i.</p

DV capsid protein induces HMGB1 release with the involvement of PCAF acetylase complex.

<p>A, K562 cells were transfected with individual plasmid encoding for DV capsid protein or GFP and stable cell line K562-Capsid and K562-GFP were obtained, respectively. IFA and Western blot were performed on the cell lysate to detect for the expression of capsid-GFP fusion protein (39 kDa) and GFP (27 kDa). The cell nuclei were stained blue with DAPI (i) and actin was used as a control to ensure equal loading of the cell lysate (ii). Cell culture supernatants of the stable cell line were harvested and concentrated to probe for the presence of HMGB1 using Western blot. B, stable cell line K562-Capsid was subjected to EP treatment and 3 days post EP treatment, the cell culture were harvested and concentrated to detect for the presence of HMGB1 using Western blot. The band intensities of HMGB1 of the EP treated cell culture supernatants were measured in relation to the band intensity of untreated control cell culture media (assigned to value of 1). C, siRNA was employed to knockdown PCAF protein in the K562-Capsid cells and cell viability assay was performed to evaluate for cytotoxicity upon siRNA transfection. D, K562-Capisd was transfected with siRNA to knockdown PCAF protein and both cell lysate and cell culture supernatants were collected 72 hrs post-transfection. Western blot was performed on the cell lysate and cell culture supernatants to detect for the PCAF and HMGB1, respectively. The band intensities of PCAF protein from the siRNA-transfected cells were compared in relation to the band intensity of untransfected control cell lysate. Actin was used as a control to ensure equal loading of total protein. The band intensities of HMGB1 of siRNA transfected cell culture media was measured in relation to the band intensity of K562-Capsid cells without siRNA. E, the amount of HMGB1 released into the cell culture supernatants from K562-Capsid cells treated with EP or transfected with 0, 25, 50 or 100 nM of siRNA were measured using ELISA. * denotes p-value <0.05 for T-tests comparing the mean amount of HMGB1 detected in EP-treated K562-Capsid cell culture supernatants to that of the untreated K562-Capsid cells. ⧫ denotes p-value <0.05 for T-tests comparing the mean amount of HMGB1 detected in siRNA transfected K562-Capsid cell culture supernatants to that of the non-siRNA transfected K562-Capsid cells.</p

DV induces translocation of HMGB1 from cell nuclei to cytoplasm and into the extracellular milieu.

<p>A, Immunofluorescence analyses (IFA) were performed on DV-infected K562. K562 cells were infected at M.O.I. 10 and fixed at 3 d.p.i. HMGB1 was stained green with rabbit anti-HMGB1 antibody and goat anti-rabbit IgG FITC conjugated antibody while DV was stained red using mouse anti-E antibody and anti-mouse-IgG Alexa Fluor 532. The cell nuclei were stained blue with DAPI. B, K562 cells were mock-infected, treated with UV-irradiated DV (UV-DV) at a M.O.I. 10, infected with DV (M.O.I. 10), or treated with 5 µg/ml of LPS. The nuclear and cytosolic fractions were harvested at 3 d.p.i. and Western blot analysis was performed to detect for HMGB1. The band intensities of HMGB1 of the mock-infected, UV-DV treated, DV-infected and LPS-stimulated nuclear fractions were expressed as a ratio to its respective cytosolic fraction. The housekeeping proteins TFIID and actin were included as loading controls for the nuclear and cytosolic fractions, respectively. C, K562 cells were infected with DV at M.O.I. of 1. Cell culture supernatants from DV-infected K562 were harvested at 3 d.p.i. and concentrated for the detection of HMGB1 via Western blot. D, DV-infected PBM cells infected at M.O.I. 1 and the cells fixed at 3 d.p.i. for IFA. HMGB1 was stained green with rabbit anti-HMGB1 antibody and goat anti-rabbit IgG FITC conjugated antibody while DV was stained red using mouse anti-E antibody and anti-mouse-IgG Alexa Fluor 532. The cell nuclei were stained blue with DAPI. E, PBM were infected with DV at a M.O.I. of 1 and Western blot analysis of HMGB1 in the cell culture supernatants of DV-infected PBM was performed. The band intensities of HMGB1 from DV-infected sample and LPS-treated cells at day 1 p.i. were assigned to a value of 1. The relative fold difference in the intensities of DV-infected samples and LPS-treated cells from day 2- to 5 p.i. was measured in relation to the band intensity its respective treatment group at day 1 p.i.</p

DV mediates HMGB1 release through the involvement of PCAF acetylase complex.

<p>A, K562 cells were transfected with 25, 50 or 100 nM of siRNA for 3 days and cell viability assay was performed to evaluation of the cytotoxicity of siRNA transfection on K562 cells. The percentage of the viable transfected cells was expressed as a percentage to that of the non-transfected cells. B, siRNA (concentration of 25 to 100 nM) targeting against PCAF was used to transfect K562 cells. The transfected K562 cells were subjected to DV infection 48 hours post-transfection. The cell lysates and culture supernatants were harvested at 1 d.p.i. and Western blot analysis was then performed to detect for the presence of PCAF and HMGB1, respectively. The PCAF band intensities of the siRNA transfected K562 cells were calculated in relation to the non-transfected mock-infected cells (assigned to a value of 1). Similarly, the band intensities of HMGB1 of the transfected cell culture supernatants were measured in relation to the band intensity of non-transfected DV-infected cell culture media (assigned to value of 1). Actin was used as a control to ensure equal loading. C, ELISA was performed to quantify the amount of HMGB1 released into the supernatants from siRNA transfected (0, 25, 50 or 100 nM of siRNA) and DV-infected K562 cells. ⧫ denotes p-value <0.05 for T-tests comparing the mean amount of HMGB1 detected in siRNA transfected K562 cell culture supernatants to that of the non-siRNA transfected K562 cells. D, K562 cells were transfected with 0, 25, 50 or 100 nM of siRNA for 48 hours before infected with DV. The supernatants were harvested at 1 d.p.i. and plaque assay was performed to quantify the viral yield.</p

A proposed model of DV capsid protein induces HMGB1 release from monocytes.

<p>DV enters the monocytes and uncoat. The capsid protein enters the nucleus and triggers the acetylation of HMGB1 by PCAF complex. HMGB1 then translocates from the nucleus to cytoplasm, a process that can be inhibited by EP. HMGB1 gets released from the monocytes and binds to RAGE receptors on endothelial cells, triggering a signalling pathway which leads to the lost of vascular integrity. The increased in vascular leakage can be repressed by HMGB1-neutraling antibody or anti-RAGE antibody.</p