Introduction {#s1}
============

The group of cellular proteins known as Interferons (IFNs) appeared to be expressed in infected cells as an early response to viral infection but, in addition to their antiviral activity, IFNs also have a profound effect on cell growth [@pone.0036909-Vannucchi1].

IFN-α2 was the first human protein shown to be effective for cancer treatment and the first economically viable clinical product developed from recombinant DNA technology in cancer therapy.

Antitumor activity of IFNs is probably exerted through direct and indirect mechanisms. It is conceivable that numerous direct effects play a central role in the overall antitumor response, such as down-regulation of oncogene expression, induction of differentiation, inhibition of cell cycle progression and induction of tumor suppressor genes, and programmed cell death [@pone.0036909-Bekisz1]. However, additional understanding of IFN antitumor action and mechanisms influencing tumor responsiveness or resistance appears necessary to aid further promising development of biomolecular strategies in IFN therapy of cancer.

The most extensively studied anticancer treatment-induced mechanism is apoptotic programmed cell death. Nevertheless, the correlation between the induction of apoptosis and drug response cannot explain the overall tumor cell sensitivity [@pone.0036909-Rebbaa1]. Numerous recent studies have shown that in cells where apoptosis is blocked, non-apoptotic cell death or irreversible cell growth arrest, namely senescence, can be activated as potential tumor-suppressor mechanisms [@pone.0036909-Kim1]. The concept of senescence is applied in general to the irreversible proliferative arrest of cells caused by various stresses [@pone.0036909-Chiantore1] including oxidative damage, telomere dysfunction, and DNA damage. One particularly relevant source of stress in tumor cells is derived from the aberrant proliferative signals of oncogenes which may trigger senescence through a process known as oncogene-induced senescence, functioning as a potential tumor suppressor mechanism.

Senescent cells are identified *in vitro* by distinctive morphological changes, such as large cell size, flat vacuolated morphology, the inability to synthesize DNA, the formation of domains of heterochromatin called Senescence-Associated Heterochromatin *Foci* (SAHF) and the expression of a Senescence-Associated β-galactosidase activity (SA-β-gal) [@pone.0036909-Campisi1], [@pone.0036909-Dimri1]. One of the earliest steps in the senescence programme is the translocation of histone chaperone HIRA (Histone Repression factor A) into promyelocytic leukemia (PML) nuclear bodies (NBs), but the role played by HIRA localization into PML bodies has not yet been identified. PML bodies are nuclear structures known to serve as sites of protein modification and the assembly of macromolecular regulatory complexes, and have been extensively implicated in the induction of senescence and apoptosis [@pone.0036909-Condemine1], [@pone.0036909-Geoffroy1].

Consistent with its role in tumor suppression, the critical senescence pathways converge on the two major tumor suppressor genes p53 and pRb, whose mutations or inactivation are most common in all cancers [@pone.0036909-Shay1].

Abrogation of senescence can be achieved by SV40 large T, a combination of HPV oncoproteins E6 and E7, E1A and MDM2 coexpression or small interfering RNA against pRb and p53 [@pone.0036909-Boehm1]. Tumors initiated by loss of p53 can be eliminated by senescence induced by p53 restoration, tumor regression being achieved through an innate immune response that leads to the clearance of senescent cells [@pone.0036909-Ventura1], [@pone.0036909-Xue1]. Cellular senescence results in altered gene expression including IFNs and their related genes [@pone.0036909-Yoon1]. PML is known to be regulated by the interferon pathway via the STAT transcription factors [@pone.0036909-Stadler1]. IFN also regulates many other components of PML nuclear bodies, suggesting that in conjunction they mediate the antiviral and antiproliferative activities of this cytokine [@pone.0036909-Geoffroy1]. Cellular senescence is induced in human fibroblasts by prolonged IFN-β treatment through DNA damage signaling and a p53-dependent pathway [@pone.0036909-Moiseeva1]. IFN-α also induces replicative senescence in endothelial cells after continuous stimulation [@pone.0036909-Pammer1]. Taken together, these studies suggest that signaling through the IFN pathway might play an important role in cellular senescence.

Human Papilloma Viruses (HPVs) are small DNA viruses involved in the development of both benign and malignant lesions localised in different anatomic districts, that are able to replicate exclusively in the stratified squamous cutaneous and mucosal epithelium. More than 100 different HPV types have been isolated so far, and they can be sub-grouped into cutaneous or mucosal according to their ability to infect the skin or the mucosa of the genital or upper-respiratory tracts [@pone.0036909-ZurHausen1]. To date, the causative association between mucosal high risk-HPV and cervical carcinoma has been clearly demonstrated. It is well known that the expression of E6 and E7 viral oncoproteins is a common feature of cervical cancer cells and is strongly implicated in the process of cancer development, E6 and E7 principally targeting and inhibiting p53 and pRb tumor suppressor proteins, respectively [@pone.0036909-Hebner1]. Emerging lines of evidence support the involvement of the cutaneous HPV types belonging to the beta genus in non melanoma skin cancer (NMSC). However, although the role of beta HPV types in NMSC in Epidermodysplasia verruciformis (EV) patients is well accepted, their involvement in skin carcinogenesis in the general population is not entirely proven. The transforming properties of the majority of cutaneous HPV types have been poorly investigated. Tommasino and co-workers [@pone.0036909-Caldeira1] have shown that HPV38 E7 appears to act similarly to HPV16 E7, by binding to pRB and promoting its degradation via the proteasome pathway. On the contrary, HPV38 E6 oncoprotein differs from mucosal high risk HPV E6 proteins in the mechanism by which it counteracts p53 activity. The expression of HPV38 E6 and E7 in human keratinocytes induces the stabilization of p53, which in turn selectively activates transcription of ΔNp73, a p53 inhibitor [@pone.0036909-Accardi1]. High ΔNp73 levels have been found in a number of human malignancies including cancers of the breast, prostate, liver, lung and thyroid [@pone.0036909-Buhlmann1]. Recently, it has been shown that IFN-α reduces ΔNp73 levels in Huh7 hepatoma cells, and this effect correlates to increased susceptibility to IFN-α triggered apoptosis [@pone.0036909-Testoni1].

Here, we show that prolonged treatment with IFN-β induces senescence in cutaneous HPV38-transformed keratinocytes. PML is essential in IFN-β induction of senescence in HPV38-transformed keratinocytes, and both p53 and p21 pathways contribute to the execution of the phenomenon. p53 colocalyzes with IFN-β-induced PML into PML Nuclear Bodies. By recruitment of p53 into NBs, IFN-β can modulate p53 post-translational modification at specific phosphorylation and acetylation sites and downregulate ΔNp73 expression, leading to the recovery of p53 transactivating activity of selected target genes involved in cell proliferation control.

Results {#s2}
=======

IFN-β Inhibits Cell Proliferation of K38 and K16 Cells {#s2a}
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Keratinocytes expressing E6 and E7 proteins of HPV-38 and HPV-16, referred to as K38 and K16 respectively, were obtained as described [@pone.0036909-Caldeira1].

To test whether IFN-β could affect proliferation of both transformed keratinocytes, K-38 and K-16 cells were treated with IFN-β for several time points and the amounts of viable cells were revealed. As shown in [Fig. 1A](#pone-0036909-g001){ref-type="fig"}, proliferation of both cell lines was strongly affected by IFN-β.

![IFN-β affects cell proliferation in K16 and K38 cells.\
(A) IFN-β inhibits cell proliferation in K16 and K38 cells. Cells were seeded in triplicate at 10^5^ cells per 35 mm dish and, after 24 h, IFN-β was added to the cultures for the indicated times. IFN-β treated and control cells were counted in a hemocytometer and viability was evaluated by trypan blue exclusion. Data represent means ± s.d. of three independent experiments. \*\* = p\<0.01; \*\*\* = p\<0.001. **(**B**)** IFN-β treatment differently affects cell cycle progression of K16 and K38 cells. K16 and K38 cells were treated with IFN-β for the indicated time points. DNA staining was performed by incubating cells in PBS containing 0.18 mg/ml propidium iodide and 0.4 mg/ml DNase-free RNase. Cells were analysed on a FACScan flow cytometer. DNA profiles, derived from one representative experiment of three performed, are shown. (C) IFN-β treatment differently affects DNA synthesis in K16 and K38 cells. To determine the number of S-phase nuclei, cells were plated in triplicate at 10^5^ cells per 35 mm dish, treated with IFN-β for different time points and incubated with 50 µM BrdU for the last 5 hours. BrdU treated samples were then fixed and stained with an anti-BrdU monoclonal antibody followed by a rhodamine conjugated goat anti-mouse antibody. BrdU-positive cells were counted under a fluorescence microscope. Data represent means ± s.d. of three independent experiments.](pone.0036909.g001){#pone-0036909-g001}

To exclude that the antiproliferative effect of IFN-β could be due to downregulation of E6/E7 expression, we checked HPV-38 and HPV-16 E6 and E7 mRNA levels by RT-PCR. No significant variations were observed in either E6 or E7 expression in K38 and K16 cells upon treatment with IFN-β for different time points (data not shown).

We previously demonstrated that IFN-β exerts its antiproliferative effect on high-risk HPV-positive cell lines by lengthening cell cycle S-phase progression [@pone.0036909-Vannucchi2]. We analyzed cell cycle distribution of K38 and K16 cells after treatment with IFN-β for several time points. Both cell lines showed a significant augment of S-phase cell amount starting from 48 h of treatment. Interestingly, in K16 cells S-phase cell accumulation increased with time whereas in K-38 cells the S-phase increase was followed by an augment of G1 population ([Fig.1B](#pone-0036909-g001){ref-type="fig"}).

K16 and K38 cells were pulse-labelled with BrdU for 5 h and analysed for BrdU incorporation. As with what was observed in SiHa and other mucosal high-risk HPV-positive cell lines [@pone.0036909-Vannucchi2], an increased number of BrdU-positive cells reflecting an S-phase cell accumulation was revealed in IFN-β treated K16 populations. On the contrary, in K-38 samples the number of cells incorporating BrdU upon IFN-β treatment appeared clearly reduced ([Fig. 1C](#pone-0036909-g001){ref-type="fig"}).

IFN-β Induces Cellular Senescence in K38 but Not in K16 Cells {#s2b}
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To study apoptosis and senescence induction, specific assays were performed. Annexin-V externalization assay showed no significant increase of apoptosis in either cell types after IFN-β treatment (data not shown). Senescent cells were quantified by counting cells displaying β-galactosidase activity at pH 6.0 (SA-βgal). This lysosomal hydrolase is elevated in senescent cells as a result of lysosomal activity at suboptimal pH, which is detectable only in senescent cells due to an increase in lysosomal content. Interestingly, increasingly high percentages of senescent cells were observed exclusively in K38 cells transformed by E6 and E7 proteins of cutaneous HPV genotype, starting from 4 days of IFN-β treatment, compared to control keratinocytes (LXSN), K16 cells and mucosal high risk HPV-positive cell lines ([Fig. 2A, B, and C](#pone-0036909-g002){ref-type="fig"}).

![IFN-β induces senescence in K38 cells.\
(A) Control keratinocytes (LXSN), K16, K38 cells and high risk HPV-positive squamous carcinoma cell lines ME-180, Caski, HeLa and SiHa were treated with IFN-β for 4 days and senescent cells were quantified by counting cells displaying senescent-associated β-galactosidase (SA-βgal) activity at pH 6.0. (B) Percentage of senescent cells in K16 and K38 cells treated with IFN-β for different time points. (C) SA-βgal-positive K38 blue cells observed under a light microscope after 4 days of IFN-β treatment. \* = p\<0.05; \*\*\* = p\<0.001.](pone.0036909.g002){#pone-0036909-g002}

Involvement of PML, p53 and p21 in Cell Senescence Induced by IFN-β in K-38 Cells {#s2c}
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It is known that important senescence regulators are found in IFN-inducible genes. In addition, it has been reported that prolonged IFN-β stimulation can induce senescence in normal cells through the activation of a DNA damage response triggered by an ATM-chk2-p53 pathway [@pone.0036909-Moiseeva1]. We asked whether IFN-β could induce the senescence phenotype in K38 cells through the involvement of PML and the activation of p53, thus counteracting the inhibitory action exerted on p53 by HPV-38 E6/E7 expression. We analysed the protein levels of PML, p53 and p21, upon IFN-β treatment, and the respective involvement in senescence through RNA silencing (siRNA) technique. Three different siRNAs were used for PML, p53 and p21 genes. Upon IFN-β treatment, PML was up-regulated as well as p21 ([Fig. 3](#pone-0036909-g003){ref-type="fig"}). On the other hand in K16 cells, even if IFN-β treatment induces PML expression, it is not detectable any increase in p21 protein levels (data not shown).

![IFN-β affects the expression of proteins involved in senescence in K38 cells.\
Western blot analysis of PML, p53 and p21 expression in K38 treated with IFN-β for different time points. Whole cell extracts were resolved on SDS-PAGE and transferred onto PVDF membrane. Immunoblotting was performed as reported in M&M.](pone.0036909.g003){#pone-0036909-g003}

PML seems to be an essential component of senescence response in K38 cells, since, when PML expression is inhibited by specific siRNAs ([Fig. 4A](#pone-0036909-g004){ref-type="fig"}), IFN-β-induced senescence is strongly reduced ([Fig. 4D](#pone-0036909-g004){ref-type="fig"}). p21 silencing ([Fig. 4C](#pone-0036909-g004){ref-type="fig"}) partially affects IFN-β-induced senescence ([Fig. 4D](#pone-0036909-g004){ref-type="fig"}), while p53 silencing ([Fig. 4B](#pone-0036909-g004){ref-type="fig"}) appears much more effective ([Fig. 4D](#pone-0036909-g004){ref-type="fig"}), suggesting that different p53 targets may be involved in IFN-β-induced senescence in K38 cells and different pathways may cooperate towards this phenomenon.

![Effect of PML, p53 and p21 silencing on senescence induction by IFN-β in K38 cells.\
PML (A), p53 (B) and p21 (C) were silenced by specific small interfering RNAs and protein expression was analyzed by Western blot in cells treated with IFN-β for 4 days. (D) Senescence induction by IFN-β (4 days treatment) was evaluated by SA-βgal staining. \* = p\<0.05; \*\* = p\<0.01.](pone.0036909.g004){#pone-0036909-g004}

PML Target Proteins Colocalyze in PML Nuclear Bodies {#s2d}
----------------------------------------------------

It is known that PML recruits into NBs p53 and different proteins involved in p53 post-translational modifications that are critical for the activation of p53 and for the selection of target genes [@pone.0036909-Bourdeau1]. We studied colocalization of PML with p53 and ΔNp73 through confocal microscopy analyses of K38 cells treated with IFN-β for different time points. [Fig. 5](#pone-0036909-g005){ref-type="fig"} shows that p53 (A) and ΔNp73 (B) colocalyze with IFN-β-induced PML into PML NBs. On the other hand, colocalization is not detectable after PML silencing (data not shown).

![p53 and ΔNp73 co-localyze with IFN-β-induced PML into PML Nuclear Bodies.\
(A,B) For confocal microscopy analysis, K38 cells were cultured on glass bottom dishes (MatTek Corporation) and treated with IFN-β for 4 days. Cells were then fixed in PBS 4% paraformaldehyde for 30 min on ice, immuno-fluorescence labelling was performed as described in [Materials and Methods](#s4){ref-type="sec"} and sample were analyzed using confocal microscope (Leica TCS SP5).](pone.0036909.g005){#pone-0036909-g005}

Post-translational Modification of p53 by IFN-β {#s2e}
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The expression of HPV38 E6 and E7 in human keratinocytes induces the stabilization of p53, as shown by WB analysis of p53 in K38 cells compared with control keratinocytes (LXSN), K16 cells and high risk HPV-positive cell lines SiHA and ME-180 ([Fig. 6A](#pone-0036909-g006){ref-type="fig"}). This p53 stabilization can be related to increased phosphorylation [@pone.0036909-Accardi1] and acetylation ([Fig. 6C](#pone-0036909-g006){ref-type="fig"}).

![IFN-β effect on p53 post-translational modification and expression of its target genes.\
(A) WB analysis of p53 in control keratinocytes (LXSN), K16 and K38 cells and in high risk HPV-positive squamous carcinoma cell lines SiHa and ME-180 treated with IFN-β for 48 h. (B) WB analysis of p53 phosphorylated at different phosphorylation sites. (C) WB analysis of acetylated p53. (D) WB analysis of phosphorylated and acetylated p53 and ΔNp73 in K38 cells silenced by PML siRNA and treated with IFN-β for 48 h. (E) WB analysis of ΔNp73 in K38 cells treated with IFN-β for different time points. Whole cell extracts were resolved on SDS-PAGE and transferred onto PVDF membrane. Immunoblotting was performed as reported in M&M. (F) Real time PCR analysis of Bax and Pig3 was carried out on K38 cells treated with IFN-β for 48 h, also in the presence of PML siRNA. NT  =  not transfected. \* = p\<0.05; \*\* = p\<0.01.](pone.0036909.g006){#pone-0036909-g006}

In this respect, we may hypothesize that IFN-β, through PML up-regulation, can lead to the recovery of p53 transactivating activity of target genes involved in cell proliferation control. Therefore p53 phosphorylation and acetylation status was analyzed in K38 cells treated with IFN-β. IFN-β modulates p53 phosphorylation status at different phosphorylation sites (Ser-6, Ser-15, Ser-46, Ser-392, [Fig. 6B](#pone-0036909-g006){ref-type="fig"} **)** while acetylation is mainly downregulated in Lys-320 ([Fig. 6C](#pone-0036909-g006){ref-type="fig"}). Ser-6, Ser-392, and Lys-320 seem to be the most important p53 post-translational modifications involved in IFN-β-induced senescence in K38 cells. In fact, when PML expression is silenced, IFN-β is not able to modulate p53 Ser-6, Ser-392, and Lys-320 status ([Fig. 6D](#pone-0036909-g006){ref-type="fig"}).

Accardi *et al*. [@pone.0036909-Accardi1] reported that p53 stabilization in K38 cells leads to transcriptional activation of ΔNp73, a p53 inhibitor, able to inhibit p53 transactivation of genes involved in cell growth suppression. It has been shown that IFN-α reduces ΔNp73 levels in Huh7 hepatoma cells and this effect correlates to increased susceptibility to IFN-α triggered apoptosis [@pone.0036909-Testoni1]. We observed that in K38 cells, IFN-β treatment downregulates ΔNp73 mRNA levels (data not shown). The ΔNp73 protein expression appears to be reduced upon IFN-β treatment ([Fig. 6E](#pone-0036909-g006){ref-type="fig"}), probably as a result of the p53 post-translational modifications induced by IFN-β. In fact, when PML expression is silenced, ΔNp73 protein levels are not downregulated by IFN-β ([Fig. 6D](#pone-0036909-g006){ref-type="fig"}).

Real time PCR array results indicate that some genes involved in senescence and growth control are IFN-β-upregulated ([Fig.6F](#pone-0036909-g006){ref-type="fig"}). In particular, the observed induction of p53 target genes Bax and Pig3 indicates that IFN-β treatment leads to the recovery of p53 transactivating activity of selected target genes involved in the control of cell proliferation. In fact, it has been reported that modification of specific p53 phosphorylation and acetylation sites may correlate to the transactivation of growth related genes, suggesting a tissue and promoter-specific p53 activity regulation [@pone.0036909-Dai1]. PML depletion reduces IFN-β induction of Bax and Pig3 in K-38 cells ([Fig.6F](#pone-0036909-g006){ref-type="fig"}), indicating the role of PML in the ability of IFN-β to recover p53 transactivation activity of specific target genes.

Discussion {#s3}
==========

IFNs were the first successful biological therapy for human malignancy and currently there are several approved IFN cancer therapies. Clinical effectiveness of different IFN subtypes in treatment of various forms of cancer has been extensively reviewed [@pone.0036909-Vannucchi1]. Better definition of therapeutic molecular targets appears to be critical to fully realize the potential of IFNs in oncology and further understand the mechanisms of antitumor action of the IFN family.

Senescence is a permanent cell cycle arrest that is resistant to growth factors and other signals that induce cell proliferation. It has been proposed that senescence prevents cancer in the early stages of its development [@pone.0036909-Campisi1]. Tumor suppressors such as p53, pRb and PML are critical regulators of senescent programme [@pone.0036909-Shay1], [@pone.0036909-Bourdeau1], and genes required for senescence are often found to be mutated in human cancers. Cellular senescence is induced in human fibroblasts by prolonged IFN-β treatment through DNA damage signaling and a p53-dependent pathway [@pone.0036909-Moiseeva1]. IFN-α also induces replicative senescence in endothelial cells after continuous stimulation [@pone.0036909-Pammer1], and treatment with IFN-γ induces cellular senescence in young human umbilical vascular endothelial cells [@pone.0036909-Kim2]. However, whether induction of senescence is sufficient to repress tumor *in vivo* is controversial. Recent reports showed that conditional restoration of p53 in mice with hepatocarcinomas, sarcoma or lymphoma is able to promote tumor regression [@pone.0036909-Xue1], [@pone.0036909-Ventura1]. In addition, it has been reported that HeLa cells cease proliferation and undergo senescence by introduction of the bovine papillomavirus E2 gene that inhibits the expression of the HPV18 E7 gene [@pone.0036909-Johung1]. Antisense sequences directed against HPV16 E6 and E7 genes transfected in SiHa cells contributed to apoptosis and senescence [@pone.0036909-Sima1].

In contrast to mucosal high-risk HPV types, the involvement of cutaneous HPV types in human carcinogenesis is still unclear. Cutaneous HPV types that belong to the beta genus of the HPV phylogenetic tree were first isolated in patients suffering from EV, a rare autosomal recessive cancer-prone genetic disorder, and are consistently detected in NMSC from EV, immunocompromised and normal individuals [@pone.0036909-Karagas1]. The transforming properties of the majority of the cutaneous HPV types have been poorly investigated. It has been reported that cutaneous HPV5 E6 protein targets and abrogates Bak function by promoting its proteolitic degradation both *in vitro* and in regenerated epithelium [@pone.0036909-Underbrink1]; however, regulation of Bax has also been reported [@pone.0036909-Struijk1]. The E6 protein of HPV5 compromises the repair of UV-induced thymine dimers [@pone.0036909-Giampieri1] and E6 of HPV7 forces keratinocytes into the S1-phase by inhibiting p53-activated, pro-apoptotic genes [@pone.0036909-Giampieri2]. HPV8 E6 is able to bind XCRR1 that functions in a single strand DNA repair [@pone.0036909-Iftner1] and it has been shown that UV-irradiated cutaneous HPV8 E2-transgenic mice develop invasive carcinomatous lesions more rapidly than non-irradiated counterparts [@pone.0036909-Pfefferle1]. Moreover, E6/E7 expression of HPV20 influences proliferation and differentiation of the skin of UV-irradiated transgenic mice [@pone.0036909-Michel1]. The anti-apoptotic activity and the delay of the DNA repair mechanism may lead to the persistence of UV-damaged keratinocytes, suggesting that cutaneous HPV types may be involved in the early stages of carcinogenesis.

A different mechanism behind the lack of cell cycle arrest in cutaneous HPV expressing cells is the up-regulation of ΔNp73 as a result of p53 accumulation observed in HPV38 E6 and E7 expressing human keratinocytes. ΔNp73 in turn inhibits the capacity of p53 to induce the transcription of genes involved in growth suppression [@pone.0036909-Caldeira1], [@pone.0036909-Accardi1]. This observation, together with the efficiency of pRb binding and degradation by HPV38 E7, the HPV38 E6/E7-induced suboptimal activation of telomerase and the HPV38 E6/E7 transforming properties *in vivo* [@pone.0036909-Gabet1], seems to indicate that HPV38 E6 and E7, differently from proteins of other cutaneous HPV types, may be involved in the maintenance of oncogenic transformation.

We have previously reported that type I IFNs inhibit cell proliferation in high risk mucosal HPV-positive Squamous Carcinoma Cell (SCC) lines by inducing a significant accumulation of cells in S-phase [@pone.0036909-Vannucchi2]. The S-phase deregulation triggers apoptotic cell death specifically mediated by the pro-apoptotic factor TRAIL [@pone.0036909-Vannucchi3].

The present study shows that IFN-β affects cell proliferation in keratinocytes expressing E6 and E7 proteins of cutaneous HPV-38 to a greater extent than in E6 and E7 mucosal HPV-16 transformed cells. In particular, K38 cells undergo senescence upon prolonged IFN-β treatment. IFN-β appears to induce senescence by up-regulating the expression of the tumor suppressor PML. Indeed, experiments of gene silencing via specific siRNAs have shown that PML is essential in the execution of senescence programme and that both p53 and p21 pathways are involved in senescence induction by IFN-β in K38 keratinocytes.

P53 and PML are critical mediators of senescence. PML is essential for the formation of discrete protein assemblages in the nucleus known as Nuclear Bodies (NBs) [@pone.0036909-Ishov1]. PML recruits into NBs p53 and proteins involved in p53 post-translational modifications that are essential for the activation of p53 and for the selection of target genes, such as the DNA damage responsive kinases ATM and ATR [@pone.0036909-Bourdeau1]. ATM kinase phosphorylates p53 at Ser-15, a senescence-inducible modification [@pone.0036909-Webley1], in IFN-β-induced cellular senescence in human fibroblasts [@pone.0036909-Moiseeva1]. Over-expression of PML is capable of inducing premature senescence by stabilizing p53 via p53 acetylation on Lys-382 and phosphorylation on Ser-15 and Ser-46 [@pone.0036909-Ferbeyre1]. In contrast, deacetylation of p53 antagonizes PML-induced premature senescence [@pone.0036909-Langley1].

It has been shown that PML interacts with CBP/p300 acetyltransferase and stabilizes p53 through Lys-382 acetylation [@pone.0036909-Ferbeyre1]. PML also recruits the tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) which induces p53Ser46 phosphorylation [@pone.0036909-Moller1]. It has been reported that HIPK2-mediated phosphorylation of p53Ser46 is required for the CBP-induced p53 acetylation at Lys-382 [@pone.0036909-Hofmann1]. PML has also been recently identified as a direct target of p53 revealing a regulatory positive feedback loop between p53 and PML [@pone.0036909-deStanchina1].

Our results indicate that in K38 cells p53 colocalyzes with IFN-β-induced PML into PML NBs. IFN-β can significantly modulate p53 phosphorylation at Ser-6,-15,-46, and -392 and acetylation status mainly at Lys-320. It has been also shown that p53 acetylation in Lys-320 is the first step in IFN-β-induced senescence in human fibroblasts [@pone.0036909-Moiseeva1].

DNA damage response is required for the activation of p53 in response to oncogenes. Oncogene induced senescence is accompained by DNA replicative stress, including prematurely terminated DNA replication forks and DNA double-strand breaks caused by hyper-DNA replication [@pone.0036909-DiMicco1]. Consistent with this, Ras-induced senescence is associated with activation of DNA damage response effectors, such as ATM/ATR and Chk2/Chk1, and inactivation of these DNA damage effectors by RNA interference attenuates oncogene induced senescence [@pone.0036909-Mallette1], [@pone.0036909-Toledo1]. We observed that the inhibition of ATM and ATR prevents IFN-β induction of senescence in K38 keratinocytes, suggesting that IFN-β might induce senescence through a p53-dependent DNA damage pathway (data not shown).

It has been reported that HPV16 E6 mediates resistance to IFN-induced senescence through inhibition of p53 acetylation by binding to CBP/p300. Conversely, treatment of HPV16 E7-expressing cells with IFN ultimately resulted in cellular senescence through a process that is dependent upon acetylation of p53 by CBP/P300 [@pone.0036909-Hebner2]. Moreover, HPV16 E7 up-regulates SIRT1, thus attenuating p53 activity via its deacetylation [@pone.0036909-Allison1]. It has been shown that HPV16 E6 can induce multiple site phosphorylation of p53 [@pone.0036909-Zhang1]. HPV38 E6 and E7 expression in human keratinocytes induces phosphorylation of p53, which leads to the up-regulation of ΔNp73 and the inhibition of p53 transcriptional induction of genes involved in growth suppression [@pone.0036909-Accardi1]. All together these observations indicate that p53 post-translational modifications are critical for p53 involvement in senescence programme induced by IFN-β and that modulation of p53 activity could be a common strategy utilized by both mucosal and cutaneous HPV to inhibit p53 function.

We report that by recruitment of p53 into NBs via PML induction, IFN-β may modulate p53 phosphorylation and acetylation status and downregulate ΔNp73 expression in K38 keratinocytes, leading to the recovery of p53 transactivating activity of selected target genes involved in cell proliferation control. Real time PCR array confirms that some genes involved in senescence and growth control are IFN-β-upregulated, in agreement with the reported observations [@pone.0036909-Bourdeau1] that modification of specific p53 phosphorylation and acetylation sites may correlate to the transactivation of growth related genes, suggesting a tissue and promoter-specific p53 activity regulation.

Our results contribute to one of the most interesting current research questions about the exact contribution of post-translational modification sites to the selectivity of the global transcriptional programme of p53. Other important questions remain to be answered to clarify the multitude and redundancy of p53 covalent post-translational modifications required for p53-dependent senescence. It is possible that no single specific post-translational modification leads to specific p53 gene transactivation activity, but each modification might help to regulate p53 function in a tissue and promoter-specific manner.

Materials and Methods {#s4}
=====================

Cell Cultures and Treatments {#s4a}
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Primary human foreskin keratinocytes were transduced with empty retrovirus pLXSN (control), or with pLXSN38E6E7 or pLXSN16E6E7 as described by Caldeira et al., 2003 [@pone.0036909-Caldeira1] and were grown in KBM BulletKit (Lonza).

HPV16-positive cell line Caski and SiHa, HPV18-positive cell line HeLa, and HPV68-positive cell line ME180, obtained from the American Type culture Collection, were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum.

Cells were maintained in a humidified atmosphere of 5.5% CO~2~ at 37°C.

Human recombinant IFN-β (Rebif; 3×10^8^ IU/mg of protein; Ares-Serono) was added to the medium at the concentration of 200 IU/ml.

Measurement of Cell Proliferation {#s4b}
---------------------------------

Transformed keratinocytes were seeded in triplicate at 10^5^ cells per 35 mm dish. After 24 h, IFN-β was added to the cultures for the indicated times. Adherent cells were detached with 0.05% trypsin-0.02% EDTA in PBS, suspended in growth medium, and counted in a hemocytometer. Viability was evaluated by trypan blue exclusion.

Flow Cytometry {#s4c}
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For cell cycle analysis, cells were fixed in 70% ice-cold ethanol for at least 30 min. DNA staining was performed by incubating cells in PBS containing 0.18 mg/ml propidium iodide and 0.4 mg/ml DNase-free RNase. Cells were analysed on a FACScan flow cytometer (Becton and Dickinson).

BrdU Incorporation {#s4d}
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To determine the number of S-phase nuclei, cells were plated in triplicate at 10^5^ cells per 35 mm dish, treated with IFN-β for different time points and incubated with 50 µM BrdU for the last 5 hours. Samples were fixed with 95% ethanol, 5% acetic acid, treated with 1.5 M HCl and stained with an anti-BrdU monoclonal antibody (Amersham) followed by a rhodamine conjugated goat anti-mouse antibody (Cappel).

Senescence-associated β-galactosidase Staining {#s4e}
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Cells were plated in 12 multi-well, 0,5×10^5^ cells per well, and treated with IFN-β at different time points. Senescent cells were quantified by counting cells displaying senescent-associated β-galactosidase activity at pH 6.0, assayed through Senescent Detection Kit (Calbiochem) following manufacturer's instruction.

Western Blot Analysis {#s4f}
---------------------

To analyse protein expression, control and IFN-β treated cells were lysed in SDS reducing sample buffer. Total cell extracts were clarified by centrifugation and boiled in the presence of 5% 2-Mercaptoethanol and 0.01% bromophenol blue. Protein concentration was determined (Bio-Rad Protein Assay) and 30 mg of total proteins were resolved on SDS-PAGE and transferred onto PVDF membrane (Amersham). The membranes were blocked with 5% skim milk dissolved in PBS-T and incubated with primary antibodies (mouse anti-p53; rabbit anti-p21 (Santa Cruz); rabbit anti-PML (Bethyl); rabbit anti-phospho-p53 Ser6, anti-phospho-p53 Ser15, anti-phospho-p53 Ser46, anti-phospho-p53 Ser392 (Cell Signaling); rabbit anti-acetyl-p53 Lys320, anti-acetyl-p53 Lys373/382 (Millipore), and anti-human β tubulin mouse IgG1 antibody (ICN), as an internal control. Immune complexes were detected with horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antiserums (ICN) followed by enhanced chemiluminescence reaction (Millipore).

PML, p53 and p21 silencing {#s4g}
--------------------------

Small interfering RNAs (siRNAs) targeted to PML, p53 and p21 were designed and validated by Qiagen and a non-silencing siRNA (Qiagen) served as control.

PML siRNAs were: 1) Sense:5′-CGUCUUUUUCGAGAGUCUGtt-3′;Antisense:5′-CAGACUCUCGAAAAAGACGtt-3′; 2)Sense:5′-CCCGCAAGACCAACAACAUtt-3′; Antisense: 5′-AUGUUGUUGGUCUUGCGGGtg-3′; 3)Sense:5′-GGGACCCUAUUGACGUUGAtt-3′; Antisense: 5′-UCAACGUCAAUAGGGUCCCtg-3′.

p53 siRNA were: 1) Sense:5′--GGAAAUUUGCGUGUGGAGUtt-3′; Antisense: 5′--CUCCACACGCAAAUUUCCtt-3′; 2) Sense: 5′-GCAUCUUAUCCGAGUGGAAtt-3′; Antisense: 5′-UUCCACUCGGAUAAGAUGCtg-3′; 3) Sense: 5′-GCAGUUAAGGGUUAGUUUAtt-3′; Antisense: 5′-UAAACUAACCCUUAACUGCaa-3′.

p21 siRNA (Dharmacon) was: Sense:5\'-GAUGGAACUUCGACUUUGUUU-3\': Antisense:5\'-PACAAAGUCGAAGUUCCAUCUU 3\'.

Shortly before transfection, 2×10^5^ cells per dish were seeded in 35 mm dishes in 1 ml of fully supplemented culture medium. siRNA was diluted in 50 µl culture medium without supplements to a final concentration of 10 nM. 3.5 µl of HiPerfect Transfection Reagent (Qiagen) were added to the diluted siRNA and mixed by vortexing. After an incubation of 10 min at room temperature, the transfection complex was added drop-wise onto the cells. 24 hrs post-transfection cells were treated with IFN-β for the indicated time points.

Immunofluorescence {#s4h}
------------------

Immunofluorescence staining of cells was performed on cells grown on Glass Bottom Culture Dishes 14 mm Microwell poly-d-lysine Coated from Mat Tek Corporation (Ashland, MA 01721 U.S.A.) and fixed with 4% formaldehyde, permeabilized with PBS/0.1% Triton-X, and stained using the following primary antibodies: anti-PML; ap53 (Santa Cruz), anti-ΔNp73 (Imgenex). Anti-mouse-Fitc (Cappel), anti-mouse-Alexa 546 (Molecular Probe \# A11030), and anti-rabbit-Alexa 610 (Molecular Probe \# A31551) were used as secondary antibodies. Sample were analyzed using confocal microscope (Leica TCS SP5). Software: LAS AF version 1.6.3 (Leica Microsystem).

Real-time PCR {#s4i}
-------------

Real-time PCR was carried out by using the MESA GREEN MasterMixes Plus, Low ROX (Eurogentec). The primer sequences are: Bax F: 5\' TTTGCT TCA GGG TTT CAT CC 3\', R: 5\' ATCCTC TGC AGC TCC ATG TT 3\'; Pig3 F: 5\' GCTTCA AAT GGC AGA AAA GC 3\', R: 5\' AACCCA TCG ACC ATC AAG AG 3\'.

We thank Roberto Gilardi for excellent editorial assistance.

**Competing Interests:**The authors have declared that no competing interests exist.

**Funding:**Research in our laboratory is currently funded in part by "Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale (PRIN 2008) del Ministero dell'Istruzione, dell'Università e della Ricerca" and Progetti Ateneo, Sapienza University, 2009 and 2011. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[^1]: Conceived and designed the experiments: MVC GF GR. Performed the experiments: MVC SV ZAP RA. Analyzed the data: MVC SV ZAP GV EA GF GR. Contributed reagents/materials/analysis tools: RA MT. Wrote the paper: MVC GF GR.


QuikTrip Founder Chester Cadieux Dies

Tuesday, March 15th 2016, 10:44 am

By: News On 6

Chester Cadieux, one of the two founders of QuikTrip convenience stores, has died.

The company says Cadieux died peacefully at his home in Tulsa Monday night. He was 84.

In a statement, the company said, "Chester's vision, keen wit, insistence on fairness, and marvelous ability to mentor people will never be forgotten."

The statement said Cadieux considered himself to be luckier than smart. He said his secret was to hire good people and promote from within.

Cadieux and Burt Holmes opened the first store in Tulsa in 1958. According to QuikTrip, Holmes came up with the idea of opening a small grocery store in Tulsa in 1957 after seeing successful 7-11 stores on a trip to Dallas. Holmes convinced his former classmate, Chester Cadieux, to invest in the store, calling it QuikTrip.

Chester's son Chet became president of QuikTrip in 2002. As of 2014, the company had 700 stores and was named to Fortune magazine's list of 100 Best Companies To Work For for the 12th straight year.

Tulsa Chamber of Commerce President Mike Neal issued the following statement on Cadieux's passing:

“Chester’s successful legacy as one of Tulsa’s most celebrated entrepreneurs is a testament to the stellar growth of the company he co-founded, QuikTrip, and its loyalty to its employees and commitment to its hometown.

We lost a true visionary business leader today, and our thoughts are with the Cadieux family and the entire QuikTrip organization.”

Learn More About Chester Cadieux's Story On John Erling's Voices Of Oklahoma


Q:

How can I disconnect clients from MySQL?

I need an efficient way to disconnect all clients with a given username from MySQL.  I thought about changing the users password but I think that is only checked when the connection is made.
Ideas?

A:

You could use "SQL to SQL" method below (just pass in extra connection options to mysql client as needed):
shell> mysql -NBe "SELECT CONCAT('KILL ', id, ';') FROM information_schema.processlist WHERE user = 'some_username';" | mysql -vv

Note: This works with MySQL 5.1 and 5.5. This would have to be implemented differently for older MySQL versions as information_schema does not have the processlist table.
Options used:
-N means that you do not want to get column names back.
-B puts it into batch mode, so that you do not get MySQL's table layout.
-e executes the following statement.
-v controls the verbosity, could be used up to three times.

Explanation of how it works:
First the KILL statements are generated along with IDs.
shell> mysql -NBe "SELECT CONCAT('KILL ', id, ';') FROM information_schema.processlist WHERE user = 'some_username';"

Sample output:
KILL 1061;
KILL 1059;
KILL 1057;

Then those statements are executed.
shell> mysql -NBe "SELECT CONCAT('KILL ', id, ';') FROM information_schema.processlist WHERE user = 'some_username';" | mysql -vv

Sample output:
--------------
KILL 1061
--------------

Query OK, 0 rows affected

--------------
KILL 1059
--------------

Query OK, 0 rows affected

--------------
KILL 1057
--------------

Query OK, 0 rows affected


F1 is developing several different onboard camera ideas and wants to introduce a shot that gives viewers a better view of what drivers can see from their crash helmets.

These developments are ongoing but include the possibility of two-time world champion Alonso, who is leaving F1 at the end of the season, trialling a new camera before the end of the season.

Alonso’s crash helmet supplier Bell has an option where the camera would be placed on the side of the helmet, level with the driver’s eyeline.

Discussions have already taken place about using Alonso to experiment with an onboard camera and Bell was keen to do something last weekend in the United States.

Fernando Alonso, McLaren Photo by: Steven Tee / LAT Images

However, it is understood that this was deemed too soon.

“We were thinking to test the camera together with Bell and FOM but at the end it didn’t happen this weekend,” said Alonso.

“I don’t know if it’s going to happen in the next weekends. It will be good to experiment.

“Maybe for the last race or something it would be a nice memory to have.”

Ensuring the output of the camera is broadcast-quality is an important factor but F1 views safety as the key parameter and there are steps it must go through if a camera is used that would interfere with the helmet itself.

While Bell’s camera would be mounted outside, F1 is understood to be working on other designs that include one that would be embedded in the internal padding of the helmet to the side of the driver’s head and level with their eyeline.

Anything that involves potentially altering the internals of the helmet needs to be greenlit by the helmet manufacturer but crucially the FIA, which is responsible for homologating designs.


% -------------------------------------------------------------------------------------------------------------------------
function net = add_adjust_layer(net, name, input, output, params, gain, bias, lr_gain, lr_bias)
% -------------------------------------------------------------------------------------------------------------------------
    net.addLayer(name, ...
                 dagnn.Conv('size', [1, 1, 1, 1]), ...
                 {input}, {output}, ...
                 params);
    filters = net.getParamIndex(params{1});
    biases = net.getParamIndex(params{2});
    net.params(filters).value = single(gain);
    net.params(biases).value = single(bias);
    net.params(filters).learningRate = lr_gain;
    net.params(biases).learningRate = lr_bias;
end


China's Alibaba restructures online retailer Taobao

Taobao, China's largest online retailer, has split into three separate companies to better address its target markets, parent company Alibaba Group said Thursday.

The move could also foreclose a public listing of Taobao, an option that Yahoo, a stakeholder in Alibaba, had hoped would lead to an increase in the value of its investment, according to an analyst.

The restructuring creates Taobao Marketplace, a consumer-to-consumer platform designed for consumers and small businesses; Taobao Mall, a business-to-consumer marketplace; and eTao, which will target the shopping search market. All three companies will continue under the Alibaba Group.

Taobao currently dominates China's online retail market with a 71.6 percent share, according to Beijing-based research firm Analysys International. The site is very popular and reported reaching 370 million users at the end of 2010.

But Taobao, which first took off as a consumer-to-consumer site in 2003, has also been working to expand its e-commerce business. The site launched Taobao Mall in 2008, as a business-to-consumer platform that now features more than 30,000 international and domestic brands available to shoppers.

In an e-mail to Alibaba employees, company CEO Jack Ma said the company is making the move as e-commerce has faced "disruptive changes," pointing to social trends and the entrance of new companies in the market. "Significant change has taken place in customer demand," he said. "We need to offer consumers more sophisticated and customized services."

In spite of Alibaba's dominance in the overall e-commerce market, Taobao only has a 31.4 percent share of China's business-to-consumer market, according to Analysys International. Numerous competitors like Chinese electronics retailer 360buy and Amazon's Chinese site are all vying for the business.

Alibaba's eTao service also faces an uphill battle in its bid to get search engine users. China's Baidu is currently the country's largest search engine with a 75.8 percent share, and many users frequent the site to seek online goods, according to analysts. Alibaba aims to attract those users to its own eTao search platform, analysts said.

Even as the restructuring will allow the new companies to better focus on their core businesses, the move is also part of Alibaba's strategy to attract other players to take advantage of its platforms, according to Ma's letter.

Alibaba has tried to market its eTao as a search engine that shows results for all e-commerce companies, including rivals, said Mark Natkin, managing director of Beijing-based Marbridge Consulting. But Alibaba's competitors have been reluctant to support it, as eTao was operated by Taobao.

"At the moment, the perception within the industry is that Taobao is a competitor rather than a facilitator, so I think companies have been a little unsure if the eTao platform has their best interests at heart," he said. But now that eTao is a company on its own, other e-commerce players may be more be willing to support the search platform, Natkin added.

Still, some analysts question the timing of Alibaba's announcement and if more might be at play. The Chinese company has been involved in a recent dispute with Yahoo, which has a 43 percent stake in Alibaba.

The dispute arose when it was revealed last month that Alibaba Group decided to transfer its successful online payment service known as Alipay to a separate company controlled by Ma. By doing so, the move has threatened to devalue Yahoo's investment in Alibaba Group.

Alibaba defended the move and said it was done in order to obtain a license from the Chinese government to operate Alipay. But Yahoo fired back, arguing that the transfer in ownership occurred without the company's knowledge, a claim that Alibaba denies.

The dispute over Alipay underscores the ongoing discontent between the two companies over business issues. Last year, Alibaba held negotiations with Yahoo to buy back its stake, but those talks went nowhere.

Yahoo, however, may feel pressured to return to the negotiating table. It has been willing to "stomach" its difficult relationship with Alibaba because of the Chinese company's growing worth, Natkin said. Many believed Alibaba would list Taobao within the next 12 to 18 months, which would only further generate value for Yahoo's investment, he added.

Thursday's announcement, however, makes that more unlikely. Instead, Ma's letter said that the company "won't rule out" the possibility of taking Alibaba Group public. As a result, investors and Yahoo will probably have to wait longer for the company to list, Natkin said.

"It could put Yahoo in a situation (where) it has to make a decision: staying in a relationship where the romance has long ago faded, or go ahead and sell back its stake now to bring a conclusion to that relationship," Natkin added.


Thermo-Quickleen

The Pies Have It ~ Aussie Cuisine?

by Tenina Holder

My Menus

After many requests and many more half started New Year's Posts (some of which will see the light of day, time permitting) this was a recipe I could get up on here quickly before the half way point of the first month of twenty ten was staring back at me. If last year was anything to go by, this year will feel like about 2 months, and I'll be adding Christmas recipes before I can finish singing Auld Lang Syne! Never one for weight loss resolutions, this will certainly not help in that department, but what the hell, Lets Get Fat this year!

Shortcrust Pastry...always easy in the Thermomix, but if you are Thermomix-less, feel free to buy or simply make by hand. Remember the colder your butter/hands/workbench the better. I have a fantastic new book Ratio by Michael Ruhlman which you should all rush out and buy...you will never need a recipe for pastry again, but for those of you without that book, this is for you;

Place flour, butter and salt into Thermo bowl and blend 3 sec/speed 6.

8

Add water through hole in lid as you continue to blend on speed 5 until pastry forms a ball. Remove from bowl and mold into flat disc. Chill whilst you make filling.

9

Roll shortcrust pastry out to 2mm thickness and line 6 large muffin holes in ‘Texas’ size muffin tin. Double line the base of each pie, by re rolling pastry and cutting to suit. Place in freezer until use.

10

Fill lined pie shells with cold mixture, top with puff pastry top. Cut cross slit in pastry and cook for 30-35 minutes until golden and puffy. Allow to cool slightly before removing from tray.

11

If you prefer to skip the whole shortcrust base thing, do so, use the ceramic pie dish (or dishes for individuals) and just use Puff Pastry as a topper...as shown. YUM!

Sous Vide Steak with Red Capsicum Puree

My Menus

Who am I?

Tenina Holder is a wife and mum and more recently grandmother, who started cooking for a living back in 2006.

Tenina has become the premium go to source for all thermomix expertise and of course fresh and easy recipes that work. Her cooking classes are sold out in literally hours, her cookbooks frequently appear on the Australian best seller lists and her Spin A Dinner app hovers around on top of the charts.

Her following is growing and global with people as far away as Chile and Kazakhstan loving her books and sending her email! Tenina is not afraid of salt, butter or sugar and believes chocolate is a health food.


/*
 * Copyright (C) 2010 Google Inc. All Rights Reserved.
 * Copyright (C) 2013 Apple Inc. All Rights Reserved.
 *
 * Redistribution and use in source and binary forms, with or without
 * modification, are permitted provided that the following conditions
 * are met:
 * 1. Redistributions of source code must retain the above copyright
 *    notice, this list of conditions and the following disclaimer.
 * 2. Redistributions in binary form must reproduce the above copyright
 *    notice, this list of conditions and the following disclaimer in the
 *    documentation and/or other materials provided with the distribution.
 *
 * THIS SOFTWARE IS PROVIDED BY APPLE COMPUTER, INC. ``AS IS'' AND ANY
 * EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE
 * IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR
 * PURPOSE ARE DISCLAIMED.  IN NO EVENT SHALL APPLE COMPUTER, INC. OR
 * CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL,
 * EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO,
 * PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR
 * PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY
 * OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT
 * (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE
 * OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.
 *
 */

#ifndef DocumentEventQueue_h
#define DocumentEventQueue_h

#include "EventQueue.h"
#include <wtf/HashSet.h>
#include <wtf/ListHashSet.h>
#include <wtf/OwnPtr.h>

namespace WebCore {

class Document;
class Event;
class Node;

class DocumentEventQueue FINAL : public EventQueue {
public:
    explicit DocumentEventQueue(Document&);
    virtual ~DocumentEventQueue();

    virtual bool enqueueEvent(PassRefPtr<Event>) OVERRIDE;
    virtual bool cancelEvent(Event&) OVERRIDE;
    virtual void close() OVERRIDE;

    void enqueueOrDispatchScrollEvent(Node&);

private:
    void pendingEventTimerFired();
    void dispatchEvent(Event&);

    class Timer;

    Document& m_document;
    OwnPtr<Timer> m_pendingEventTimer;
    ListHashSet<RefPtr<Event>, 16> m_queuedEvents;
    HashSet<Node*> m_nodesWithQueuedScrollEvents;
    bool m_isClosed;
};

}

#endif // DocumentEventQueue_h


655 S.W.2d 654 (1983)
Virginia R. McCONNELL, Plaintiff-Respondent,
v.
ST. LOUIS COUNTY, Missouri and Robert E. Emerick, Defendants-Appellants.
No. 44512.
Missouri Court of Appeals, Eastern District, En Banc.
June 14, 1983.
Motion for Rehearing and/or Transfer Denied August 1, 1983.
Application to Transfer Denied September 20, 1983.
*655 Ben Ely, Jr., Victoria Spann Sheehan, St. Louis, for defendants-appellants.
Rene E. Lusser, St. Louis, for plaintiff-respondent.
Motion for Rehearing and/or Transfer to Supreme Court Denied August 1, 1983.
SMITH, Presiding Judge.
Defendant appeals from a judgment against it of $65,000 based on a jury verdict. The judgment was affirmed by a panel of this court and a motion for rehearing thereafter granted before an expanded panel. We now reverse the judgment in part.
The lawsuit arose from a one-bus accident occurring during a "Fall Foliage" outing sponsored by the defendant, St. Louis County. Mrs. McConnell was one of 40 persons who made claims as a result of the accident. She subsequently died from causes unrelated to the accident and her executrix was substituted as plaintiff. Her suit was brought against defendant and Robert Emerick, the bus driver, who was discharged in bankruptcy prior to trial.[1] Defendant confessed liability and offered to confess judgment for $20,300.90 which was rejected by plaintiff. That figure constituted the amount remaining of defendant's $800,000 liability insurance policy not already distributed to other claimants through settlement. The amount remaining was stipulated to by the parties.
On appeal defendant raises two issues. The first is that the judgment should have been entered for $20,300.90 because of the restrictions of Sec. 537.610, RSMo 1978, imposing a maximum liability of $800,000 on St. Louis County. The second point involves the correctness of a damage instruction. Because we agree with defendant's first contention and because it offered before trial to confess judgment for $20,300.90 we need not reach the second contention.
Plaintiff contends that Secs. 537.600 and 537.610 are not applicable here because the accident occurred while the county was exercising a proprietary rather than a governmental function.
In Jones v. State Highway Commission, 557 S.W.2d 225 (Mo. banc 1977), the Supreme Court abolished the doctrine of sovereign immunity prospectively for torts occurring "on and after August 15, 1978." The General Assembly responded to that decision by enacting Secs. 537.600-537.650. Sec. 537.600 provides that: "Such sovereign or governmental tort immunity as existed at common law in this state prior to September 12, 1977 [the date of Jones, supra], except to the extent waived, abrogated or *656 modified by statutes in effect prior to that date, shall remain in full force and effect;...." Prior to Jones, the courts held that municipalities were not protected by sovereign immunity for torts arising from their proprietary functions but were protected from such torts arising from their governmental functions. This distinction was not applicable to the state and its political subdivisions which were fully protected under the immunity doctrine for their tortious acts. Wood v. County of Jackson, 463 S.W.2d 834 (Mo.1971) [1, 2]; Payne v. County of Jackson, 484 S.W.2d 483 (Mo.1972). We specifically so held as to St. Louis County in Coleman v. McNary, 549 S.W.2d 568 (Mo.App.1977). See also, Connor v. Crawford County, 588 S.W.2d 532 (Mo.App.1979). Pre-Jones, school districts were treated as political subdivisions for immunity purposes. Rennie v. Belleview School District, 521 S.W.2d 423 (Mo. banc 1975) [2]; Smith v. Consolidated School District No. 2, 408 S.W.2d 50 (Mo. banc 1966) [4-6]. The governmental-proprietary distinction drawn as to municipal corporations, but not as to political subdivisions, created the anomalous situation that a plaintiff's right of recovery for the same tortious conduct depended on whether the tortfeasor was a municipality or a political subdivision such as a county. See, Wood v. County of Jackson, supra (Finch, J., Concurring). Nevertheless, the governmental-proprietary distinction was not applied pre-Jones to political subdivisions of the state.
Following passage of 537.600-537.610, the case of State ex rel. Allen v. Barker, 581 S.W.2d 818 (Mo. banc 1979), came before the court. That case involved an activity which could arguably fit either the governmental or the proprietary function as defined in municipality cases. The entity involved, however, was a school district, where theretofore the nature of the function made no difference. The court, seizing upon the language of Sec. 537.600, concluded that the reinstatement of pre-Jones law served to reinstate also the governmental-proprietary dichotomy. The court did not address the line of pre-Jones cases holding such dichotomy of function was inapplicable when determining immunity of political subdivisions other than municipalities. The court in Barker remanded the case to give the plaintiff an opportunity to amend the petition "to aver sufficient facts to bring the cause within the [proprietary] exception to the general doctrine of immunity." Barker does not specifically overrule any of the pre-Jones cases. Following Barker, three appellate decisions in reliance upon Barker have concluded that the immunity of a school district is to be determined with reference to the governmental-proprietary distinction. Allen v. Salina Broadcasting, Inc., 630 S.W.2d 225 (Mo.App.1982); Johnson v. Carthell, 631 S.W.2d 923 (Mo.App. 1982); Fowler v. Board of Regents, etc., 637 S.W.2d 352 (Mo.App.1982).
From Barker, Allen, Johnson and Fowler, plaintiff argues that all sovereign immunity is now determined by reference to the governmental-proprietary function test. We cannot interpret those cases so broadly. At most they serve to bring school districts into the fold with municipalities. But in the absence of an express statement that such a test is also to apply to counties, or an express overruling of the pre-Jones cases regarding counties, we are unable to conclude that Barker and its progeny intended such a sweeping result. We find it especially difficult to reach such a conclusion in view of the statutory language reinstating sovereign immunity as it existed prior to Jones. Prior to that decision, St. Louis County was immune from the suit brought by the plaintiff and we do not find that Barker changes that immunity. Plaintiff's right of recovery rests upon Sec. 537.600 and .610 to which we now turn.
Sec. 537.600 creates two situations in which sovereign immunity is waived, i.e.: (1) operation of motor vehicles and (2) condition of property. Plaintiff's cause of action is clearly within the first situation. In Bartley v. Special School District of St. Louis County, 649 S.W.2d 864 (Mo. banc 1983), the Supreme Court held that in addition to those situations the public entity must have purchased liability insurance (or establish a self-insurance plan) for the *657 waiver to be effective. That was also done here. The limits of plaintiff's recovery against St. Louis County must be determined by reference, therefore, to Sec. 537.610.
In pertinent part that Section provides:
"(1) ... but the maximum amount of such coverage shall not exceed eight hundred thousand dollars for all claims arising out of a single occurrence ... and no amount in excess of the above limits shall be awarded or settled upon. Sovereign immunity for the state of Missouri and its political subdivisions is waived only to the maximum amount of and only for the purposes covered by such policy of insurance....
(2) The liability of the state and its public entities on claims within the scope of sections 537.600 to 537.650 shall not exceed eight hundred thousand dollars for all claims arising out of a single accident or occurrence ...." (Emphasis supplied)
In our divisional opinion we held that determination of the amount that plaintiff could recover on this judgment was premature and that resolution of that issue should await attempts to collect the judgment. Upon reconsideration it appears that such a holding overlooks the doctrine barring collateral attack on a judgment and the language of Sec. 537.610. As was stated in Hammett v. Hatton, 189 Mo.App. 567, 176 S.W. 1078 (1915) [1]:
"... validity of a judgment cannot be impeached on a motion to quash an execution issued on it. Such a motion cannot be based on the ground of mere error or irregularity in the judgment. The jurisdiction not being questioned, and the judgment not having been reversed, vacated or set aside, such a motion would constitute a collateral attack on it."
A motion to quash an execution cannot be substituted for an appeal and a defense which might have been valid in the underlying proceeding cannot be raised in a collateral proceeding such as a motion to quash execution. First National Bank in Chester v. Connor, 485 S.W.2d 667 (Mo.App. 1972) [2-6].[2]
It is also clear from Sec. 537.610 that no amount in excess of the statutory limit may be "awarded or settled upon." The statute does not speak to collection, but rather to award. It further provides that immunity is waived only to the extent of the limits set forth in the statute and that is the extent of the liability of the public entity. This language indicates to us that the judgment entered must conform to the statutory limits imposed by the General Assembly. Had this claim been the only one made for this accident and had the verdict exceeded the $100,000 one-claimant limit we would have no doubt that the judgment to be entered would have to have been $100,000. We can see no difference where the parties have stipulated that the amount already paid in settlement brings the jury award above the statutory limit. While the judgment normally follows the verdict, this is not inevitably true. Where the amount of recovery is limited as a matter of law, the judgment of the court must reflect that limitation regardless of the verdict. See, for example, MAI 4.11, Notes on Use 2; Parker v. Pan American World Airways, Inc., 447 S.W.2d 731 (Tex.Civ.App.1969) [1, 2]. The liability of the County under the statute was limited to $20,300.90 as a matter of law and the judgment of the court should have been for that amount.
We address two additional issues raised by plaintiff. The first is a contention that she is entitled to a pro rata share *658 of the amount of her verdict equivalent to the percentage that $800,000 bears to the total amount of settlements plus her verdict. This contention is based upon 537.610.4. That subparagraph provides an optional procedure which is available to any party. It does not impose any mandatory requirement upon the public entity to seek apportionment. Nor could such an apportionment proceeding increase the liability of the public entity beyond the limit statutorily established. If that subparagraph is available to give plaintiff an additional recovery, that recovery cannot come from defendant.[3]
Plaintiff also contends the judgment should be left intact to protect her rights against the County's insurer for bad faith settlement and disbursement of the $800,000. Normally, bad faith refusal to settle (which plaintiff claims here) creates a cause of action by the insured against the insurer who has subjected the insured to liability beyond the limits of the policy. That, of course, is not the situation here. The County's liability is limited to $800,000 and exhaustion of the insurance proceeds does not render it liable for the overage. Some courts have recognized an obligation of the insurance company to deal fairly with all claimants and have allowed suits directly against the insurer by claimants in situations not unlike this one where allegations of bad faith settlement are made. See, Haas v. Mid America Fire & Marine Ins. Co., 35 Ill.App.3d 993, 343 N.E.2d 36 (1976); Alford v. Textile Ins. Co., 248 N.C. 224, 103 S.E.2d 8 (1958); Bartlett v. Travelers' Ins. Co., 117 Conn. 147, 167 A. 180 (1933). We do not reach the availability or merits of such an action here. Plaintiff's verdict is a matter of record, and we are unable to see how our reduction of the judgment against the county to the limits established by law can preclude plaintiff from establishing the amount of her loss under such a theory. We, of course, do not intimate that such bad faith exists, we merely recognize plaintiff's contention that it does.
Judgment reversed and cause remanded with directions to enter judgment for plaintiff and against defendant St. Louis County for $20,300.90.
REINHARD, STEPHAN, SNYDER, SATZ, SIMON and KAROHL, JJ., concur.
PUDLOWSKI, J., dissents.
KELLY, J., joins in dissent.
PUDLOWSKI, Judge, dissenting.
I respectfully dissent for the reasons set forth in my previous authored opinion hereinafter stated. I would affirm the circuit court's judgment.
The relevant facts are as follows: On October 12, 1978, plaintiff-respondent Virginia McConnell was one of approximately 43 passengers of a St. Louis County bus as part of the Fall Color Tour. Ms. McConnell, then aged 79 years and an active lady, suffered severe personal injuries when the bus overturned upon leaving the roadway. She sustained fractured ribs, a collapsed lung, a fracture of the right humerus, a pelvic fracture, and a fracture of the right clavicle. For several months following the accident, she underwent rehabilitation and therapy for these injuries. On November 21, 1979, Ms. McConnell died, for reasons unrelated to the bus accident. Virginia Klamon, her daughter and executrix of her estate, was substituted as nominal plaintiff on April 17, 1980.
Respondent's claim was one of 40 claims brought against St. Louis County and Robert E. Emerick, the driver of the bus, who has since been discharged under bankruptcy. The claims were brought under RSMo § 537.600. Under the statute the common law governmental tort immunity is effective except for limited exceptions: injuries *659 arising from the negligent acts or omissions by public employees arising out of the operation of motor vehicles (§ 537.600(1)), and injuries caused by the condition of the public entity's property if the property was in a dangerous condition at the time of the injury (§ 537.600(2)).
According to § 537.610, the total extent of a public entity's liability "shall not exceed eight hundred thousand dollars for all claims arising out of a single accident or occurrence and shall not exceed one hundred thousand dollars for any one person in a single accident or occurrence, except [under Missouri Workmen's Compensation Law]." Appellant St. Louis County's insurance carrier, The Hartford Accident and Indemnity Company, made settlements with the 39 other claimants from the bus accident, leaving a balance of $20,300.90 with which to reimburse the last claimant, Virginia McConnell.
At trial, the jury awarded Ms. McConnell $65,000. Appellant St. Louis County contends that the trial judge erred in failing to set aside the judgment of $65,000 and enter a judgment in the amount of $20,300.90 because appellant alleges that St. Louis County could not be liable beyond the $800,000.00 for all claims arising out of this single accident. I disagree and would affirm the judgment of the trial court.
The trial court did not err in refusing to set aside the jury award of $65,000. Appellant's argument as to liability is premature. As I understand the statute, the public entity may be subject in situations similar to this set of facts to money judgments and claims in excess of $800,000 but is responsible for payment of $800,000. Under the sovereign immunity statute, § 537.610(4) clearly contemplates and makes provisions for the possibility that the total amount of the awards and settlements chargeable against the County may exceed $800,000. The statute clearly provides that an apportionment, or pro-rata distribution may be made if the total sum of awards and settlements exceeds such amount. Section 547.610(4) provides in full:
If the amount awarded to or settled upon multiple claimants exceeds eight hundred thousand dollars, any party may apply to any circuit court to apportion to each claimant his proper share of the total amount limited by subsection 1 of this section. The share apportioned each claimant shall be in the proportion that the ratio of the award or settlement made to him bears to the aggregate awards and settlements for all claims arising out of the accident or occurrence, but the share shall not exceed one hundred thousand dollars. (emphasis added).
Respondent claims that she is entitled to her proportion as set forth in this statute. Respondent contends that a reduction of the judgment from $65,000 to $20,300.90, which is the residue of the $800,000, would deny her the right to a fair proportionate share as provided under this section of the statute. I need not address the issue of the appropriate dollar value to be placed on respondent's claim under the statute because that issue of collection and the issue of enforcement of this judgment are not presently before us.
Appellant also alleges that the court erred in submitting plaintiff's verdict directing Instruction No. 4, M.A.I. 31.07 because it was misleading and confusing. I disagree. Taking all the instructions as a whole, I do not find that the jury was mislead or confused in their instructions to adequately compensate the deceased Virginia McConnell for any damages she sustained as to the accident. I would dispose of this point pursuant to Rule 84.16(b). I find no error of law and an extended opinion on this point would have no precedential value.
Therefore, I would affirm the decision of the trial court as to the validity of the judgment and as to the appropriateness of the jury instruction.
NOTES
[1]  Both Emerick and St. Louis County have appealed. On appeal Emerick challenges only the court's damage instruction. Emerick was retained in the suit for "orderly prosecution" of the cause. We do not find it necessary to address the instruction question solely because Emerick is a named appellant.
[2]  Some cases have indicated that such collateral attacks are available where the record "affirmatively discloses that the judgment is void" (McDougal v. McDougal, 279 S.W.2d 731 (Mo. App.1955) [31-33]) or partially void (Kennedy v. Boden, 241 Mo.App. 86, 231 S.W.2d 862 (1950) [3, 4]). We are not convinced that the stipulation in the record of the amounts paid to other claimants is the necessary affirmative disclosure in the record that the judgment is partially void sufficient to support a collateral attack. Nor does it serve judicial efficiency to avoid the issue on direct appeal of the judgment and force it to be litigated collaterally on execution of the judgment.
[3]  We need not decide whether that subparagraph affords any practical remedy in cases such as this. The language is in the past tense indicating that apportionment cannot be sought until after awards have been made or settlements arrived at. We entertain serious doubts that such a procedure could be utilized to require payment back from claimants who have contractually settled their claims, thereby infringing on those contracts which in many cases involve compromise of anticipated judgments.


Synthesis and preliminary pharmacological studies on dihydropyrido-1,3,4-triazepinone derivatives.
New 4,5-dihydropyrido-[2,3-e]-1,3,4-triazepin-5-one derivatives (2-9) were synthesized. The preliminary pharmacological tests revealed antinociceptive action of compounds 4-7 and 9 and antianxiety action of compound 4.


// Copyright (c) 2012 The Chromium Authors. All rights reserved.
// Use of this source code is governed by a BSD-style license that can be
// found in the LICENSE file.

#include "ppapi/shared_impl/ppapi_switches.h"

namespace switches {

// Enables the testing interface for PPAPI.
const char kEnablePepperTesting[] = "enable-pepper-testing";

// Specifies throttling time in milliseconds for PpapiHostMsg_Keepalive IPCs.
const char kPpapiKeepAliveThrottle[] = "ppapi-keep-alive-throttle";

}  // namespace switches


I say "people", not "athletes" because Taylor is much more than a phenomenal running back and stellar athlete, he is a person who cares and respects the game of football, his peers, and carries himself in an unselfish and professional manner that is almost unique in today’s "there is an I in team" attitude.

The NFL is filled with middle-of-the-road players like Adam Jones who have no problem taking what they have for granted and can’t stay out of trouble.

There is also the unique case of Michael Vick, who is arguably one of the best athletes in NFL history and who made a terrible off-field decision which landed him in prison, bankrupt, and the face of dog fighting in America.

It isn't uncommon for veteran players to sit out of voluntary mini-camps and summer workouts because of their age and experience.

It is understandable that when players begin to age, staying healthy can be more of a chore than when they were young. The threat of a career ending injury during a preseason drill becomes much more real towards the end of a players career.

Unfortunately, it is also common to see players sitting out of these drills because they simply don't think they need to attend. They believe the team is lucky to have signed them, and they will perform when they are ready to perform.

Family issues, age, and veteran status aside, we all know players will stay away from camp for ridiculous reasons every year. A player who wants a larger salary, bigger bonus, longer contract extension, etc., will put all of these issues above the team.

So why is Fred Taylor, an 11-year veteran of the NFL who has over 11,000 rushing yards and over 12,000 total yards from scrimmage, acting the polar opposite of this “superstar” attitude?

The answer is simple, Fred is the rare breed of NFL athlete who can appreciate where he is, and what he does.

Taylor showed up ready to learn at New England's preseason workouts. “I decided to come out here with the rookies and the other guys returning to the team. It’s fun getting to know my teammates, getting to know all the different faces around here."

Taylor's attitude and outlook toward his new team and situation should be the blueprint for NFL players, regardless if you are the new hotshot rookie, or the veteran who has been proving himself for years.

Showing an effort, desire, and eagerness to meet your new team and striving to become a productive member of an organization are aspects of a player that are just as important for coaches to consider as age or accomplishments.

Take a look at the 2005 NFL draft. Adam "Pacman" Jones was picked sixth overall by the Titans. He was involved in several incidents (according to the Titans organization) immediately following the draft, and then skipped most of the preseason workouts because he was holding out over "contract issues."

Jones had a decent rookie season, he didn't make the Pro Bowl but he still put up respectable numbers. The Titans spent the rest of their time with Jones trying to calm him down and work with him so he would stop being involved in off-field issues.

After Jones was suspended for a year, he was traded to Dallas where his issues continued until he finally became the main event on Pros vs. Joes.

Fast forward to the second round of the 2005 NFL draft where Justin Miller, a cornerback out of Clemson, was picked up by the New York Jets.

Miller didn't have all the dazzling statistics at Clemson that Jones had at West Virginia; however, Miller never had any major character issues in college or immediately following the draft.

Miller went on to make the Pro Bowl in 2006, and after a season-ending knee injury was traded to the Raiders. Once in Oakland, Miller didn't hold out, and didn’t get into trouble.

Instead, he spent his time working out with the team and rehabbing his knee, and in 2008 had back-to-back games with kickoff returns for over 90 yards earning himself Special Teams Player of the Month for December, 2008.

The Vick situation is as big a headache as it is unique. Vick has more athletic ability than most people have ever seen which is why it gives fans of the NFL such a headache that he could throw it away so easily.

Anyone who appreciates athleticism can say they enjoyed watching Vick run all over defenses, and his arm strength is amazing (although his accuracy needs some work).

Vick didn't get the slap on the wrist that Jones did for fighting other humans and being involved in altercations that involved handguns.

Vick had the book thrown in his face and spent a good chunk of time in a federal prison for dog fighting.

Not to mention becoming the poster boy for animal cruelty in a society where dog fighting happens all the time, just ask Clinton Portis.

As far as athletic ability, Adam Jones was an average (if that) defensive back and return man who couldn't play anything other than those two positions, while Vick's athletic ability is questioned by no one and he could theoretically play running back, receiver, and quarterback.

Taylor is a phenomenal running back, but he has remained out of trouble and is getting ready to win another championship.

Adam Jones, more than likely, will never play in the NFL again, let alone start. He has been in the same sort of trouble over and over again and continues to deny involvement and give sob-story explanations for why he was even at the location of the incidents.

Michael Vick took the blame, and even though he didn't expect to have the book thrown at him, he accepted the consequences for his actions.

It's sad that the NFL and its fans have to deal with the ignorance and disregard of some players. Adam Jones will likely never play on an NFL field again.

Michael Vick absolutely deserves another shot at the NFL and has already proved himself as a worthy athlete; however, the dog fighting issue (regardless of how he accepted his punishment) was a major step in the wrong direction and resulted in him sitting out what could have been two of the most productive seasons in his career.

Fred Taylor is handling his transition to his first new team since signing with the Jaguars like a true professional and should be a role model for the Joneses and Vicks of the league.

Not only will his athleticism help the Patriots win games, his attitude will help bond a locker room and keep morale high as New England tries to take back its crown as the dynasty of the decade.