From: Subject: =?Windows-1252?Q?Neural_Stem_Cell_Tropism_to_Glioma:_Critical_Role_of_Tum?= =?Windows-1252?Q?or_Hypoxia_=97_Mol_Cancer_Res?= Date: Thu, 1 Jul 2010 23:06:59 +0200 MIME-Version: 1.0 Content-Type: multipart/related; type="text/html"; boundary="----=_NextPart_000_0000_01CB1972.1BCFA3F0" X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.5931 This is a multi-part message in MIME format. ------=_NextPart_000_0000_01CB1972.1BCFA3F0 Content-Type: text/html; charset="utf-8" Content-Transfer-Encoding: quoted-printable Content-Location: http://mcr.aacrjournals.org/content/6/12/1819.long =EF=BB=BF Neural Stem Cell = Tropism to Glioma: Critical Role of Tumor Hypoxia =E2=80=94 Mol Cancer = Res
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Neural Stem Cell Tropism to Glioma: Critical = Role of=20 Tumor Hypoxia

  1. Donghong=20 Zhao1,=20
  2. Joseph=20 Najbauer1,=20
  3. Elizabeth=20 Garcia1,=20
  4. Marianne=20 Z. Metz1,=20
  5. Margarita=20 Gutova1,=20
  6. Carlotta=20 A. Glackin3,=20
  7. Seung=20 U. Kim45 and=20
  8. Karen=20 S. Aboody12=20
  1. Divisions of 1Hematology and = Hematopoietic=20 Cell Transplantation, 2Neurosciences, and = 3Molecular Medicine, City of Hope National = Medical=20 Center and Beckman Research Institute, Duarte, California; 4Division of Neurology, Department of = Medicine, UBC=20 Hospital, University of British Columbia, Vancouver, British Columbia, = Canada;=20 and 5Institute for Regenerative Medicine, = Gachon=20 University Gil Hospital, Inchon, Korea
  1. Donghong Zhao, Division of=20 Hematology/Hematopoietic Cell Transplantation, City of Hope National = Medical=20 Center and Beckman Research Institute, 1500 East Duarte Road, Duarte, = CA=20 91010-3000. Phone: 626-359-8111, ext. 65637; Fax: 626-930-5416. = E-mail: dozhao{at}coh.org = or Karen S.=20 Aboody, Divisions of Hematology/Hematopoietic Cell Transplantation and = Neurosciences, City of Hope National Medical Center and Beckman = Research=20 Institute, 1500 East Duarte Road, Duarte, CA 91010-3000. Phone: = 626-471-7177;=20 Fax: 626-301-8857. E-mail: kaboody{at}coh.org
N= ext=20 Section

Abstract

Hypoxia is a critical aspect of the microenvironment in = glioma and=20 generally signifies unfavorable clinical outcome. Effective targeting of = hypoxic=20 areas in gliomas remains a significant therapeutic challenge. New = therapeutic=20 platforms using neural stem cells (NSC) for tumor-targeted drug delivery = show=20 promise in treatment of cancers that are refractory to traditional = therapies.=20 However, the molecular mechanisms of NSC targeting to hypoxic tumor = areas are=20 not well understood. Therefore, we investigated the role of hypoxia in = directed=20 migration of NSCs to glioma and identified the specific signaling = molecules=20 involved. Our data showed that hypoxia caused increased migration of = human=20 HB1.F3 NSCs to U251 human glioma-conditioned medium in vitro. = In HB1.F3=20 NSCs, hypoxia led to up-regulation of CXCR4, urokinase-type plasminogen=20 activator receptor (uPAR), vascular endothelial growth factor receptor 2 = (VEGFR2), and c-Met receptors. Function-inhibiting antibodies to these = receptors=20 inhibited the migration of HB1.F3 cells to glioma-conditioned medium. = Small=20 interfering RNA knockdown of hypoxia-inducible factor-1=CE=B1 in glioma = cells blocked=20 the hypoxia-induced migration of NSCs, which was due to decreased = expression of=20 stromal cell=E2=80=93derived factor-1 (SDF-1), uPA, and VEGF in glioma = cells. Our in=20 vivo data provided direct evidence that NSCs preferentially = distributed to=20 hypoxic areas inside intracranial glioma xenografts, as detected by = pimonidazole=20 hypoxia probe, as well as to the tumor edge, and that both areas = displayed high=20 SDF-1 expression. These observations indicate that hypoxia is a key = factor in=20 determining NSC tropism to glioma and that SDF-1/CXCR4, uPA/uPAR, = VEGF/VEGFR2,=20 and hepatocyte growth factor/c-Met signaling pathways mediate increased=20 NSC-to-glioma tropism under hypoxia. These results have significant = implications=20 for development of stem cell=E2=80=93mediated tumor-selective gene = therapies. (Mol=20 Cancer Res 2008;6(12):1819=E2=80=9329)

Keywords:=20
Previous=20 SectionN= ext=20 Section

Introduction

Malignant gliomas (e.g., glioblastoma multiforme) are the = most common=20 subtype of primary brain tumors, and glioblastoma multiforme is = uniformly fatal=20 with a mean survival of less than 1 year from diagnosis (1).= Hypoxia=20 is a critical aspect of the microenvironment in glioma and generally = signifies=20 unfavorable clinical outcome (2).= Current=20 surgical, radiation, and chemotherapy treatments do not alleviate the = poor=20 prognosis of this highly aggressive form of brain cancer, and effective=20 targeting of hypoxic areas in gliomas remains a significant therapeutic=20 challenge (2).= Recent=20 research indicates that neural stem cells (NSC) can specifically target = invasive=20 solid tumors, including glioma, and thus provide a novel platform for = targeted=20 delivery of therapeutic agents to tumors, resulting in significant = antitumor=20 effect (3, = 4).= However,=20 it is unknown whether NSCs are able to target the hypoxic areas in = glioma and=20 which factors induced by hypoxia may be involved in NSC-glioma tropism. = Here, we=20 investigate the role of hypoxia and identify the cytokines responsible = for=20 directed migration of NSCs to gliomas.

NSC migration toward intracranial malignant tumors was first = shown by=20 Aboody et al. (5) = and=20 Benedetti et al. (6).= They=20 showed that immortalized murine and rat NSC lines could migrate great = distances=20 to sites of orthotopic human glioma xenografts in experimental animals, = when=20 injected intracranially or i.v., and that such NSCs could deliver = anticancer=20 therapeutics to produce a significant tumor-killing effect (5, = 6).= Our=20 recent data show that human NSCs also have the ability to target = multiple tumor=20 types and exert a therapeutic effect in melanoma brain metastases (7),= =20 disseminated neuroblastoma (8, = 9),= =20 medulloblastoma (10= ), and=20 glioma (11= ). This=20 suggests that NSCs provide an efficient platform for targeted delivery = and=20 dissemination of therapeutic bioactive agents to tumors and achieve a=20 significant antitumor response.

Trafficking of stem cells to sites of injury in ischemia = models is=20 regulated by hypoxia through hypoxia-inducible factor-1=CE=B1 = (HIF-1=CE=B1; ref. 12= ).=20 Similar to tissue injury, gliomas are often characterized by profound = hypoxia=20 that leads to (a) up-regulation of HIF-1=CE=B1 (13= ) and=20 (b) overexpression of numerous cytokines and chemoattractants,=20 including hepatocyte growth factor (HGF), stem cell factor (SCF), = stromal=20 cell=E2=80=93derived factor-1 (SDF-1), urokinase-type plasminogen = activator (uPA), and=20 vascular endothelial growth factor (VEGF; refs. 14= -17= ).=20 Therefore, we hypothesized that cytokines induced by glioma-associated = hypoxia=20 may act as signals for attraction of NSCs. First, we compared the in = vitro migration of HB1.F3 NSCs toward U251 human glioma = cell=E2=80=93conditioned=20 medium under hypoxic and normoxic conditions. We then determined the = effect of=20 hypoxia on the cytokine expression profile of U251 glioma cells and the=20 corresponding receptors on HB1.F3 NSCs. Further, we showed that small=20 interfering RNA (siRNA) knockdown of HIF-1=CE=B1 blocks hypoxia-induced = migration of=20 NSCs. Our in vivo studies showed that NSCs preferentially = localized to=20 hypoxic areas in the primary tumor bed as well as the tumor border = region of=20 glioma xenografts. These data suggest that hypoxia is one of the = significant=20 factors in determining stem cell tropism to glioma.

P= revious=20 SectionN= ext=20 Section

Results

Hypoxia Induces Increased Migration of HB1.F3 NSCs

To investigate the role of hypoxia in the directed migration = of HB1.F3=20 NSCs to glioma cells, we did in vitro Boyden chamber cell = migration=20 assays. Conditioned media were collected from U251 glioma cells under = normoxia,=20 hypoxia, or after treatment with desferrioxamine mesylate (DFX). DFX is = an iron=20 chelator, which increases the levels of HIF-1=CE=B1 protein by promoting = protein=20 stability, without any effect on HIF-1=CE=B1 mRNA level, and it can be = used to mimic=20 hypoxic conditions (18= , 19= ).

We first investigated the migration of normoxic NSCs to = conditioned=20 medium from normoxic, hypoxic, or DFX-treated U251 glioma cells (Fig. = 1 ).=20 Normoxic HB1.F3 NSCs displayed highest migration to DFX-conditioned = medium (23%=20 of total cells), followed by migration to hypoxic-conditioned medium = (18%), and=20 least migration to normoxic-conditioned medium (9%). To further = investigate the=20 role of hypoxia in migration of NSCs to glioma cells, we preexposed the = HB1.F3=20 NSCs themselves to hypoxia for 24 hours and allowed the NSCs to migrate = to=20 various conditioned media from U251 cells (normoxic, hypoxic, or DFX = treated).=20 We observed significantly increased migration of hypoxic NSCs to all = conditioned=20 medium when compared with normoxic NSCs (>100% increase of NSC = migration to=20 U251 normoxic-conditioned medium, 50% increase to U251 = hypoxic-conditioned=20 medium, and 40% increase to medium from DFX-treated U251 cells; Fig. = 1). These=20 data indicate that hypoxia leads to increased migration of NSCs to = glioma cells=20 when U251 target cells only, or NSCs only, or both cell types are = exposed to=20 hypoxia, with the latter condition showing the highest NSC migration. =

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FIGURE = 1.=20

Hypoxia induces increased migration of = HB1.F3 NSCs.=20 HB1.F3 cells were preexposed to normoxia or hypoxia for 24 h. Modified = Boyden=20 chamber assays were used to determine the migration of HB1.F3 cells to = different=20 conditioned media. HB1.F3 cells (1.5 =C3=97 104) were added = to the top=20 chambers. The NSC migration assays were done under normoxia or hypoxia, = matched=20 to the normoxic or hypoxic preexposure conditions of NSCs. The number of = migrated NSCs was quantified by CyQuant fluorescence cytometry. Columns, = average=20 of cell migration in triplicate wells; bars, SD. *, P < = 0.05,=20 compared with normoxic cultures. Similar data were obtained in three = independent=20 experiments.

Effect of Hypoxia on Cytokine Expression in Glioma Cells

To identify factors that are secreted by tumor cells and = contribute to=20 NSC-tumor tropism, we used quantitative real-time PCR to screen = normoxic,=20 hypoxic, or DFX-treated U251 cells (0- to 30-hour culture) for mRNA = expression=20 of various cytokines. As shown in Fig. = 2A , we=20 detected up-regulation of SDF-1 and uPA mRNA in U251 cells following = exposure to=20 hypoxia or treatment with DFX when compared with U251 cells exposed to = normoxia=20 (18- and 24-hour culture), with peak expression levels at 24 hours after = treatment. The HGF mRNA level increased =E2=88=BC20% at 18 hours after = hypoxia or DFX=20 treatment. The expression of SCF mRNA under hypoxia and DFX treatment = increased=20 =E2=88=BC50% at 6 hours, =E2=88=BC20% at 12 hours, followed by a = decrease at 18 hours, when=20 compared with normoxia. However, expression of VEGF mRNA showed a = continued=20 up-regulation with increasing time of hypoxia or DFX treatment = (=E2=88=BC10-fold or=20 =E2=88=BC12-fold increase at 30 hours) when compared with normoxia. = Thus, both hypoxia=20 and DFX treatment resulted in up-regulation of SCF, SDF-1, uPA, and VEGF = mRNA in=20 a time-dependent manner (Fig. = 2A).=20 Because most cytokines that we investigated showed a significant = increase at 24=20 hours of hypoxia or DFX treatment, we chose 24-hour treatment of U251 = cells to=20 collect conditioned medium for our cytokine antibody array and NSC = migration=20 experiments. The antibody array data showed that levels of secreted = SDF-1 and=20 VEGF were up-regulated in U251-conditioned medium collected after = hypoxia or DFX=20 treatment (Table = 1 ; Fig. 2B = and C).=20 No significant differences were observed in the expression of HGF and = SCF as a=20 result of normoxia, hypoxia, or DFX treatment of U251 cells. uPA protein = level,=20 as measured by ELISA, increased =E2=88=BC60% after hypoxia or DFX = treatment when=20 compared with normoxia condition (Fig. = 2D).

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FIGURE = 2.=20

Hypoxia up-regulates the expression of = cytokine=20 mRNAs and proteins in U251 cells. A. Hypoxia increases = the=20 expression of cytokine mRNAs in U251 cells. U251 cells were serum = starved and=20 exposed to normoxia, hypoxia, or DFX treatment for 6, 12, 18, 24, or 30 = h=20 followed by extraction of total RNA. Real-time reverse transcription-PCR = (RT-PCR) was used to assess mRNA expression of HGF, SCF, SDF-1, uPA, and = VEGF.=20 B. Cytokine protein profiles of U251-conditioned medium = collected after exposure to normoxia, hypoxia, or DFX for 24 h as = detected by=20 Cytokine Antibody Array. C. Plotted data display the = relative=20 protein levels of HGF, SCF, SDF-1, and VEGF in U251-conditioned medium = from=20 normoxia, hypoxia, or DFX-treated cultures. SDF-1 and VEGF protein = levels were=20 increased after hypoxia or DFX treatment when compared with = normoxia-conditioned=20 medium. D. Increased level of uPA protein in = U251-conditioned=20 medium after hypoxia or DFX treatment measured by ELISA assay is shown = compared=20 with normoxia condition. Columns, mean value of triplicate wells; bars, = SD.=20 Similar data were obtained in three independent experiments. *, = P <=20 0.05; **, P < 0.01.

View this table:
Table=20 1.=20

Map of the Cytokine-Specific = Antibodies=20 Spotted onto RayBio Cytokine Antibody Array V Protein Chip=20 (RayBiotech)

Effect of Hypoxia on Expression of Cytokine Receptors in HB1.F3 = NSCs

Because cytokines mediate the migration of NSCs to glioma = cells by=20 binding to their corresponding receptors on NSCs, we investigated = whether=20 hypoxia can induce changes in the expression of cytokine receptors in = HB1.F3=20 NSCs. We determined the levels of the following receptors: c-Met = (receptor for=20 HGF), c-Kit (SCF), CXCR4 (SDF-1), uPAR (uPA), and VEGFR2 (VEGF). We = found=20 significantly greater mRNA expression for all receptors investigated, = except for=20 c-Kit, after exposure of NSCs to hypoxia or DFX when compared with = normoxic=20 condition (Fig. = 3A ). At=20 the protein level, flow cytometry data showed increased expression of = c-Met,=20 CXCR4, uPAR, and VEGFR2, but no change in c-Kit, after exposure of NSCs = to=20 hypoxia (Fig. = 3B).

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FIGURE = 3.=20

Hypoxia up-regulates the expression of = cytokine=20 receptor mRNAs and proteins in NSCs. A. Hypoxia = up-regulates=20 the expression of receptor mRNAs in NSCs. HB1.F3 cells were preexposed = to=20 hypoxia for 24 h followed by real-time RT-PCR for mRNA expression of = c-Met,=20 c-Kit, CXCR4, uPAR, and VEGFR2. Columns, mean of triplicate wells; bars, = SD.=20 Similar data were obtained in three independent experiments. *, = P <=20 0.01; **, P < 0.001. B. Hypoxia = up-regulates=20 expression of receptors in NSCs as detected by flow cytometry. Open = histograms,=20 negative control with isotype-matched antibody; solid histograms, marker = expression. In each histogram, we showed the percentage of positive = cells. The=20 data are plotted as fluorescence intensity on a logarithmic scale on the = abscissas versus the number of positive cells on the ordinates. One=20 representative figure is shown from three experiments.

Hypoxia-Induced Migration of HB1.F3 Stem Cells Can Be Blocked by=20 Neutralizing Antibodies

To determine whether HGF/c-Met, SDF-1/CXCR4, uPA/uPAR, and=20 VEGF/VEGFR2 signaling molecules were necessary for NSC tropism to = glioma, we=20 used function-blocking antibodies against SDF-1 ligand and antibodies = against=20 receptors c-Met, CXCR4, uPAR, and VEGFR2 in cell migration assays. = Anti=E2=80=93SDF-1=20 antibody inhibited the migration of NSCs to U251 normoxia- and=20 hypoxia-conditioned medium in a dose-dependent manner (10-40% and 20-60% = for=20 normoxia and hypoxia, respectively; Fig. = 4A). The=20 neutralizing antibodies to receptors c-Met, CXCR4, uPAR, and VEGFR2 = inhibited=20 the migration of NSCs by 30% to 60% and 60% to 80% in normoxic and = hypoxic=20 U251-conditioned medium, respectively (Fig. = 4B ).=20 These data further support the role of the above signaling molecules in=20 determining the tropism of NSCs to glioma.

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FIGURE = 4.=20

Hypoxia-induced migration of HB1.F3 = NSCs is blocked=20 by function-inhibiting antibodies. A. Hypoxia-induced = migration=20 of HB1.F3 NSCs is blocked by SDF-1 function-inhibiting antibody. = Conditioned=20 media from normoxic or hypoxic U251 cells were preincubated with SDF-1=20 function-inhibiting antibody for 2 h at room temperature, and then = HB1.F3 NSCs=20 were allowed to migrate to these conditioned media. B.=20 Hypoxia-induced migration of HB1.F3 NSCs is blocked by cytokine receptor = function-inhibiting antibodies. HB1.F3 NSCs were preincubated with = receptor=20 function-inhibiting antibodies or isotype-matched control antibodies for = 30 min=20 at room temperature, and then the cells were allowed to migrate to = conditioned=20 medium from normoxic or hypoxic U251 cells. The number of migrated NSCs = was=20 quantified by CyQuant fluorescence cytometry. Columns, average number of = migrated cells in triplicate wells; bars, SD. *, P < 0.01, = compared=20 with normoxic-conditioned medium. Similar data were obtained in three=20 independent experiments.

Silencing of HIF-1=CE=B1 in Glioma Cells Blocks the Hypoxia-Induced = Migration of=20 NSCs

We did siRNA-mediated knockdown of the transcription factor = HIF-1=CE=B1 to=20 examine whether HIF-1=CE=B1 was involved in the increased migration of = NSCs to glioma=20 cells under hypoxic conditions. Transfection of U251 cells with = HIF-1=CE=B1=E2=80=93specific=20 siRNA led to decreased levels of HIF-1=CE=B1 mRNA (80% reduction) when = compared with=20 control (scrambled siRNA or untransfected U251 cells; Fig. = 5A ). We=20 did Western blotting to determine the HIF-1=CE=B1 protein levels of = normoxic,=20 hypoxic, or DFX-treated U251 cells after siRNA transfection. HIF-1=CE=B1 = siRNA=20 transfection resulted in reduced amounts of HIF-1=CE=B1 protein in U251 = cells under=20 all conditions (normoxia, hypoxia, or DFX; Fig. = 5B).

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FIGURE = 5.=20

Silencing of HIF-1=CE=B1 in glioma = cells blocks the=20 hypoxia-induced migration of HB1.F3 NSCs. A. = HIF-1=CE=B1 siRNA=20 resulted in knockdown of HIF-1=CE=B1 mRNA expression in U251 cells as = detected by=20 quantitative real-time RT-PCR. B. HIF-1=CE=B1 siRNA = blocked HIF-1=CE=B1=20 protein expression in U251 cells. U251 glioma cells were transfected = with HIF-1=CE=B1=20 siRNA and treated with normoxia (N), hypoxia (H), or = DFX.=20 Western blotting was done to detect HIF-1=CE=B1 protein expression. = Full-length blots=20 are presented in Supplementary Fig. S1. C. HIF-1=CE=B1 = siRNA blocks=20 the hypoxia-induced cytokine mRNA expression. U251 glioma cells were = transfected=20 with HIF-1=CE=B1 siRNA and treated with normoxia, hypoxia, or DFX. Total = RNAs from=20 these cells were analyzed for cytokine mRNA expression by quantitative = real-time=20 RT-PCR. Columns, normalized fold cytokine mRNA expression (triplicates) = in=20 HIF-1=CE=B1 siRNA-treated cells; bars, SD. *, P < 0.01, = compared with=20 control siRNA-treated cells. D. HIF-1=CE=B1 siRNA = blocks the=20 hypoxia-induced migration of HB1.F3 cells. U251 cells were transfected = with=20 control or HIF-1=CE=B1 siRNA followed by additional incubation under = normoxia or=20 hypoxia for 24 h and collection of conditioned medium. Migration of = HB1.F3 cells=20 to conditioned medium was tested in Boyden chamber assays. Columns, = average of=20 cell migration in triplicate wells; bars, SD. *, P < 0.05, = compared=20 with conditioned medium derived from control siRNA-treated U251 cells. =

We then investigated the effect of HIF-1=CE=B1 siRNA on the = expression of=20 cytokines in U251 cells under normoxic and hypoxic conditions. = HIF-1=CE=B1 siRNA=20 inhibited the hypoxia-induced expression of SDF-1, uPA, and VEGF mRNAs, = but it=20 did not affect the cytokine levels in normoxic U251 cells (Fig. = 5C). This=20 suggests that hypoxia-induced expression of these cytokines is under the = control=20 of HIF-1=CE=B1, whereas the cytokines present in normoxic cells are = expressed=20 independently of HIF-1=CE=B1.

To further investigate the function of HIF-1=CE=B1 in NSC = migration to=20 glioma, we did NSC migration assays with conditioned medium from = normoxic and=20 hypoxic U251 cells transfected with HIF-1=CE=B1 siRNA. We found that = siRNA-induced=20 silencing of HIF-1=CE=B1 expression in U251 cells resulted in = significantly decreased=20 hypoxia-induced NSC migration (Fig. = 5D). As=20 expected based on the cytokine levels (Fig. = 5C),=20 HIF-1=CE=B1 siRNA did not affect the migration of NSCs to conditioned = medium from=20 normoxic U251 cells (Fig. = 5D). Taken=20 together, these data suggest that HIF-1=CE=B1 is a key factor in = hypoxia-induced NSC=20 migration to glioma cells by affecting the expression of multiple = cytokines in=20 U251 cells.

NSC Targeting of Glioma In vivo

To investigate the role of hypoxia in NSC tropism to glioma = in=20 vivo, U251 glioma cells were implanted into the brain of nude mice. = Two=20 months after implantation of tumor cells,=20 chloromethylbenzamido-1,1=E2=80=B2-dioctadecyl-3,3,3=E2=80=B2,3=E2=80=B2-= tetramethylindocarbocyanine=20 perchlorate (CM-DiI)=E2=80=93labeled HB1.F3 NSCs were injected = stereotactically into the=20 hemisphere contralateral to the tumor. Four days after injection of = NSCs,=20 pimonidazole was administered i.p. 20 minutes before euthanasia of the = mice.=20 Pimonidazole has proven to be an effective marker for hypoxia in = multiple tumor=20 types (20= ).=20 Immunohistochemical analysis revealed that the U251 glioma implants = contained=20 hypoxic regions that were defined by pimonidazole staining (Fig. = 6B ).=20 Within the tumor, we observed considerable numbers of NSCs associated = with the=20 hypoxic areas, with fewer NSCs detected in the normoxic areas of the = tumor (Fig. = 6B). The=20 tumor edge and the adjacent brain parenchyma also contained large = numbers of=20 CM-DiI=E2=80=93labeled NSCs (Fig. = 6C).=20 Further, the tumor edge and the hypoxic areas within the tumor were = associated=20 with high levels of SDF-1 (Fig. = 6C). SDF-1=20 may be produced by reactive astrocytes that are present around gliomas = (21= , 22= ).=20 Because HB1.F3 NSCs were injected into the contralateral normal brain=20 parenchyma, NSCs can be expected to enter into the tumor bed through the = tumor-brain interface. Indeed, we detected the largest numbers of NSCs = at the=20 tumor edge as well as in the hypoxic tumor areas in which many cytokines = are=20 up-regulated, including SDF-1. Collectively, these findings suggest that = the=20 tumor edge and the hypoxic regions in glioma are preferentially targeted = by=20 NSCs.

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FIGURE = 6.=20

NSC targeting of glioma in = vivo.=20 A. 4=E2=80=B2,6-Diamidino-2-phenylindole staining for = whole brain.=20 Dotted line, location of glioma (Tu). B. = Distribution=20 of CM-DiI=E2=80=93labeled HB1.F3 NSCs (red) in the hypoxic = (outlined by=20 broken lines) and normoxic areas of glioma xenograft, as = detected by=20 Hypoxyprobe-1 (green). Right, merged image of = CM-DiI=E2=80=93labeled NSCs and=20 Hypoxyprobe-1 staining. Scale bar, 100 =CE=BCm. C. = Localization of=20 CM-DiI=E2=80=93labeled HB1.F3 NSCs (red) to SDF-1=E2=80=93rich = regions (green)=20 in hypoxic tumor areas (dashed lines) and the tumor edge = (dotted=20 lines). Right, merged image of CM-DiI=E2=80=93labeled NSCs and = SDF-1 staining.=20 Scale bar, 100 =CE=BCm. D. Average number of migrated = NSCs in the=20 normoxic and hypoxic areas and at tumor edge as quantified by = fluorescent=20 imaging of CM-DiI signal (red). P < 0.01, compared = with=20 normoxic areas.

P= revious=20 Section= Next=20 Section

Discussion

We have previously shown the inherent tumor-tropic property = of NSCs=20 and their use as cellular vehicles for effective delivery of therapeutic = agents=20 to invasive tumors, including glioma, melanoma brain metastases,=20 medulloblastoma, and disseminated neuroblastoma. Their abilities to = track=20 infiltrating tumor cells and localize to distant tumor microfoci make = NSCs=20 attractive gene therapy vehicles with promising clinical potential (3, = 4).= A major=20 obstacle for current glioma therapies is ineffective delivery of drugs,=20 especially to hypoxic tumor areas, which are not well vascularized, = resulting in=20 limited drug penetration (23= ). Here,=20 we investigated whether HB1.F3 NSCs can target such = =E2=80=9Chard-to-reach=E2=80=9D hypoxic=20 areas in glioma, and if so, which cytokines are responsible for the NSC=20 targeting. Identification of hypoxia-induced cytokines involved in NSC = tropism=20 will be important for the development of optimal NSC-based glioma = therapies.=20

A limited number of stem cell attractants and cytokines = emanating=20 from solid tumors, including glioma, have been identified (24= -27= ).=20 However, the cytokines produced by hypoxic areas in glioma, which are=20 responsible for NSC attraction, had not been defined. It was also = unknown which=20 receptors on the surface of NSCs mediate the targeting of hypoxic = regions of=20 glioma. Here, we provide direct evidence, for the first time, that NSCs = display=20 increased migration to hypoxic glioma cells in vitro and that = NSCs=20 preferentially target the hypoxic glioma regions in vivo. Of = the=20 cytokines investigated, we found that hypoxia resulted in up-regulation = of=20 SDF-1, uPA, and VEGF mRNA and protein in U251 cells. Furthermore, the=20 corresponding receptors of these cytokines were increased on HB1.F3 NSCs = under=20 hypoxia. Importantly, by using function-inhibiting antibodies, we showed = a=20 significant reduction of hypoxia-induced NSC migration to glioma. Figure = 7=20 depicts a schematic model of NSC targeting to glioma and the cytokines = involved.=20 As mentioned above, SDF-1, uPA, and VEGF, as well as HGF, are secreted = by=20 glioma. The highest levels of these cytokines are likely produced in the = hypoxic=20 tumor regions. In addition to glioma cells, reactive astrocytes and = other=20 stromal cells located in and around the tumor may also release multiple=20 cytokines. The important role of SDF-1/CXCR4 in the migration of NSCs = has been=20 shown in models of ischemic brain injury, where SDF-1 is up-regulated in = reactive astrocytes (28= ).=20 Further, hypoxia-activated rat astrocytes display increased attraction = of neural=20 progenitor cells in vitro, and this effect is mediated by VEGF, = SCF,=20 SDF-1, and MCP-1 (29= ). These=20 findings, together with our data, suggest that a combined effect of = cytokines=20 released by hypoxic tumor and by reactive astrocytes at the tumor edge = mediates=20 attraction of NSCs. The HGF/c-Met, SDF-1/CXCR4, uPA/uPAR, and = VEGF/VEGFR2=20 ligand/receptor pairs have been implicated in glioma progression, = invasion, and=20 angiogenesis (14= , 17= , 30= ).=20 Understanding the role of these cytokines and receptors in NSC-to-glioma = targeting will help in designing exogenous NSCs with overexpression of = optimal=20 cytokine receptor combination, allowing highly effective NSC migration = and drug=20 delivery to hypoxic tumors. Our cytokine array data suggest that = multiple=20 cytokines other than the ones investigated here are also expressed in = glioma=20 cell=E2=80=93conditioned medium (e.g., RANTES, IFN-=CE=B3, TGF-=CE=B1, = and TGF-=CE=B2; Table = 1; Fig. = 2B).=20 Further work will be needed to determine the role of these cytokines in=20 NSC-to-glioma tropism.

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FIGURE = 7.=20

Schematic model of NSC-to-glioma = targeting.=20 Numerous cytokines, including HGF, SDF-1, uPA, and VEGF, are secreted by = glioma.=20 High levels of these cytokines are likely produced in the hypoxic tumor = regions.=20 In addition to glioma cells, reactive astrocytes and other stromal cells = located=20 in and around the tumor may also release cytokines. The cytokine = gradients are=20 sensed by NSCs, which then migrate to the glioma. Large numbers of NSCs = are=20 observed in the hypoxic tumor regions as well as at the tumor edge. = Note: the=20 relative amounts of various cytokines indicated in the schematic figure = may not=20 reflect the actual in vivo cytokine levels.

HIF-1 is a transcription factor that plays a crucial role = in cellular=20 responses to hypoxia in normal and pathologic tissues, including tumors = (31= , 32= ).=20 Hypoxic stimulation leads to the accumulation of HIF-1=CE=B1 protein, = which interacts=20 with HIF-1=CE=B2 and translocates to the nucleus, resulting in = transcription of=20 downstream target genes. More than 100 putative target genes for HIF-1 = have been=20 identified, including many genes involved in cell survival, migration, = invasion,=20 and angiogenesis (31= , 33= , 34= ). Among=20 gliomas, there is a strong correlation between HIF-1=CE=B1 expression, = tumor grade,=20 and tumor vascularization (31= , 35= ). HIF-1=CE=B1=20 expression is highest in the hypoxic areas immediately adjacent to = necrotic foci=20 in a subpopulation of glioma cells known as pseudopalisading cells (36= ). Our=20 data show that siRNA-mediated knockdown of HIF-1=CE=B1 in glioma cells = led to=20 significant inhibition of hypoxia-induced NSC tropism, which was due to=20 decreased expression of multiple cytokines in glioma cells. This = suggests that=20 HIF-1=CE=B1 is a key factor in hypoxia-induced NSC targeting to glioma. = Because HIF-1=20 is a =E2=80=9Cmaster regulator=E2=80=9D of many genes involved in = tumors, it will be an=20 important target for NSC-based glioma therapies.

The hypoxic environment in solid tumors renders tumor cells = more=20 resistant to radiation and chemotherapy, which correlates with tumor = recurrence=20 and poor patient prognosis (31= , 37= ).=20 Hypoxia has been identified as a major factor in radiation resistance of = glioma=20 because the low oxygen tension in hypoxic cells prevents the = radiation-induced=20 DNA damage from becoming permanent (38= , 39= ).=20 Hypoxic cells are also resistant to most chemotherapeutic drugs (37= ). The=20 reasons for such resistance include the distance of hypoxic cells from = blood=20 vessels, poor diffusion of drugs, and up-regulation of multidrug = resistance=20 genes (23= , 40= ). Given=20 the central role of hypoxia in resistance of tumors to chemotherapy and=20 radiation, targeting of hypoxic tumor cells is an attractive therapeutic = strategy (31= , 41= ). There=20 are several clinical trials aimed at overcoming treatment failure = because of=20 tumor hypoxia (41= , 42= ). We=20 expect that use of NSCs for direct delivery of anticancer drugs to = hypoxic=20 glioma will allow maximal concentrations of therapeutics at these sites, = resulting in eradication of resistant hypoxic tumor cells.

P= revious=20 Section= Next=20 Section

Materials and Methods

HB1.F3 NSC Line

The HB1.F3 NSC line was derived from telencephalon of a = human fetus=20 of 15 wk of gestation and was immortalized using a retrovirus encoding = the=20 v-myc gene (43= , 44= ). The=20 primary cells were obtained in accordance with the Guidelines of the = Anatomical=20 Pathology Department of Vancouver General Hospital, with permission to = use fetal=20 tissue granted by the Clinical Research Screening Committee Involving = Human=20 Subjects of the University of British Columbia. The HB1.F3 NSC line is a = well-characterized, clonal cell line, which is nontumorigenic and = multipotent in=20 that cells from this line can be induced to differentiate into neurons,=20 oligodendrocytes, and astrocytes (24= , 45= , 46= ). HB1.F3=20 NSCs were grown in DMEM with 10% fetal bovine serum (FBS) at 37=C2=B0C = in a=20 humidified atmosphere of 6% CO2.

Hypoxia Treatment

HB1.F3 and U251 cells were cultured to a density of = =E2=88=BC80% confluence=20 in 10% FBS DMEM and then transferred to starvation medium (2% FBS for = HB1.F3=20 cells and 0% FBS for U251 cells). The cells were then exposed to = normoxia=20 (ambient oxygen tension), hypoxia (1% O2), or DFX (150 = =CE=BCmol/L)=20 treatment for 24 h in a HEPA incubator (Thermo Forma). DFX was used as a = hypoxia-mimicking agent.

Conditioned Medium

U251 human glioma cells (American Type Culture Collection) = were=20 maintained as adherent cultures in DMEM supplemented with 10% FBS at = 37=C2=B0C in=20 humidified atmosphere containing 6% CO2. To obtain = conditioned=20 medium, U251 cells were grown on 100-mm plates to =E2=88=BC80% = confluence. Cultures were=20 rinsed thrice with serum-free medium and incubated for another 24 h in=20 serum-free DMEM. Conditioned media were collected, cleared by = centrifugation (10=20 min, 600 =C3=97 g), and stored at =E2=88=9280=C2=B0C.

In vitro Boyden Chamber Cell Migration Assay

HB1.F3 NSCs were maintained in normoxic conditions or = preexposed to=20 hypoxia for 24 h. All migration assays were done using 96-well Boyden = chambers=20 (Neuroprobe). Lower wells were filled with either U251-conditioned = medium=20 (serum-free) or DMEM as a control. HB1.F3 cells (1.5 =C3=97 = 104) were=20 added to the top chambers. After 4 h at 37=C2=B0C under normoxic or = hypoxic=20 conditions (matched to the NSC preexposure conditions), the cells that = migrated=20 to the lower membrane surface were detached by accutase solution = (Innovative=20 Cell Technologies) and transferred to a V-bottomed 96-well plate. After=20 centrifugation, migrated cells were stained using CyQuant GR fluorescent = dye=20 (Chemicon) and counted with a fluorescence microplate reader (Molecular=20 Devices). All experimental conditions were replicated a minimum of three = times,=20 and all experiments were done at least three times.

siRNA Knockdown of HIF-1=CE=B1

HIF-1=CE=B1 siRNA was purchased from Santa Cruz = Biotechnology. The HPRT-S1=20 siRNA, which served as positive control, and scrambled siRNA, which = served as=20 negative control, were purchased from IDT, Inc. U251 glioma cells were=20 transfected with HIF-1=CE=B1 siRNA using X-tremeGENE siRNA transfection = reagent=20 (Roche). The medium containing the transfection complexes was replaced = with=20 fresh medium and the cells were incubated under normoxic or hypoxic = conditions,=20 starting at 24 h after transfection. The cell migration assays, = real-time PCR,=20 and Western blotting were done 48 h after siRNA transfection.

Quantitative Real-time Reverse Transcription-PCR

Total RNA was extracted after cells were grown under = normoxic=20 conditions or preexposed to hypoxia or DFX. The RNeasy Mini kit (Qiagen) = was=20 used to isolate total RNA from HB1.F3 and U251 glioma cells according to = the=20 manufacturer's instructions. Random primers, ThermoScript reverse = transcriptase=20 (Invitrogen), and 1 =CE=BCg of total RNA were used for cDNA synthesis. = Relative=20 quantification of mRNA expression was done with the SYBR Green PCR = Master Mix=20 using an ABI PRISM 7700 thermal cycler (Applied Biosystems). The primer=20 sequences for the genes of interest are listed in Table = 2 . Each=20 sample was normalized to actin as an endogenous reference. Relative=20 quantification of target gene expression was calculated by the = comparative=20 threshold cycle (Ct) method.

View this table:
Table=20 2.=20

Primers for Quantitative = Real-time=20 RT-PCR

Measurement of Cytokine Protein Level

Cytokine Antibody Array V protein chip was used for = measurement of=20 cytokine protein levels in U251 glioma-conditioned medium according to = the=20 manufacturer's instructions (RayBiotech). Cytokine array membranes were = blocked=20 by incubation with the blocking buffer at room temperature for 30 min = and=20 incubated with 1 mL of U251-conditioned medium at 4=C2=B0C overnight. = Membranes were=20 washed thrice with Wash Buffer I and twice with Wash Buffer II at room=20 temperature for 5 min per wash and incubated with biotin-conjugated = antibodies=20 at room temperature for 2 h. Finally, the membranes were washed, = incubated with=20 horseradish peroxidase=E2=80=93conjugated streptavidin at room = temperature for 2 h and=20 with detection buffer for 2 min, and exposed to X-ray film for 30 s = (Kodak,=20 Inc.). The exposed films were digitized and the relative cytokine levels = were=20 compared after densitometry analysis using NIH ImageJ 1.36b software. = Total=20 intensity of signal spots was calculated by multiplying the area with = mean=20 signal intensity of the spot. The relative protein levels were obtained = by=20 subtracting the background signal and normalizing to the total mean = intensity of=20 the positive controls on the same membrane. Level of uPA protein in=20 U251-conditioned medium was measured using IMUBIND uPA ELISA kit = according to=20 the manufacturer's protocol (American Diagnostica, Inc.).

Flow Cytometry Assay

Cells from each cell line were resuspended in ice-cold = staining=20 buffer (PBS supplemented with 2% FBS) and incubated with Fc-block = solution for 5=20 min at 4=C2=B0C. Subsequently, cells were stained with fluorescein=20 phycoerythrin-conjugated antibodies against CXCR4, c-Kit, c-Met, uPAR, = VEGFR2,=20 or the appropriate isotype control (R&D Systems) at 4=C2=B0C for 30 = min, after=20 which cells were washed twice with staining buffer. Samples were = analyzed on a=20 Guava EasyCyte flow cytometer (Guava Biotechnologies), and the data were = processed with FlowJo7 software.

In vivo Tumor Tropism Studies

Tropism of the human HB1.F3 NSCs to orthotopic human = glioblastoma=20 xenografts was shown by implanting tumor cells (U251) into the brains of = 8-wk-old female athymic nude mice. Animals were anesthetized (132 mg/kg = ketamine=20 and 8.8 mg/kg xylazine) and received stereotactically guided injections = of 2 =C3=97=20 105 tumor cells in 2 =CE=BCL of PBS through a 30-gauge = Hamilton syringe=20 into the right forebrain (5, = 7).= Two=20 months after tumor cell injection, HB1.F3 NSCs labeled with CM-DiI = (Molecular=20 Probes) were stereotactically injected into the hemisphere contralateral = to the=20 tumor using the same coordinates. After an additional 4 d, the hypoxic = marker=20 Hypoxyprobe-1 (pimonidazole; Chemicon) was injected i.p. (60 mg/kg, 20 = min=20 before euthanasia), and then mice were perfused intracardially with 4%=20 paraformaldehyde in PBS. The brains were harvested and the Hypoxyprobe-1 = kit was=20 used for detection of hypoxia in tumors. The brains were also processed = for=20 immunohistochemical detection of various cellular/tissue antigens.

Immunohistochemistry

For immunohistochemical staining of tissues, standard = protocols were=20 done using primary antibodies against SDF-1 (R&D Systems), diluted = 1:50 (10=20 =CE=BCg/mL final concentration) in blocking buffer (5% bovine serum = albumin, 3%=20 normal horse serum, 0.1% Triton X-100). Secondary staining was done = using=20 biotinylated anti-mouse antibodies (2 =CE=BCg/mL in blocking buffer; = Vector) followed=20 by detection with avidin-FITC (1 =CE=BCg/mL). Adjacent tissue sections = were stained=20 with anti-pimonidazole antibody from the Hypoxyprobe-1 kit (1:200 = dilution in=20 PBS) overnight at 4=C2=B0C followed by staining with a FITC-conjugated = secondary=20 antibody (Chemicon). Fluorescence microscopy was done using a Nikon = TE2000-U=20 fluorescence microscope at 488 nm (FITC) and 568 nm (CM-DiI). Images = were=20 captured and processed using SPOT 4.0 (Diagnostic Instruments, Inc.). = The=20 numbers of CM-DiI=E2=80=93labeled NSCs in hypoxic and normoxic areas and = at the tumor=20 edge were counted in three mouse brains (five sections per brain). The = sizes of=20 the relevant areas (square millimeter) were determined using NIH ImageJ=20 software.

Western Blot Analysis

Total U251 cell lysate was separated in an 8% = SDS-polyacrylamide gel=20 and transferred onto polyvinylidene difluoride membranes (Millipore). = Membranes=20 were blocked for 1 h at room temperature and then incubated overnight at = 4=C2=B0C=20 with anti-HIF-1=CE=B1 antibody (Santa Cruz Biotechnology) diluted 1:200 = in 5% nonfat=20 milk in Tween 20-TBS. Blots were then stained with horseradish=20 peroxidase=E2=80=93conjugated anti-mouse secondary antibody (Santa Cruz = Biotechnology)=20 followed by enhanced chemiluminescence for signal detection = (Perkin-Elmer Life=20 Sciences).

Statistics

All data were analyzed with GraphPad Prism 4.0 software. = Data were=20 presented as mean =C2=B1 SD. Statistical differences between two groups = were=20 evaluated using two-way ANOVA. P values of <0.05 were = considered=20 significant.

= Previous=20 SectionN= ext=20 Section

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were = disclosed.

= Previous=20 SectionNext=20 Section

Acknowledgments

We thank Dr. Keely L. Walker for editing the = manuscript.

P= revious=20 SectionNext=20 Section

Footnotes

  • Grant support: AACR-National Brain Tumor = Foundation Fellowship (in memory of Bonnie Brooks); Joel A. Gingras,=20 Jr./American Brain Tumor Association Fellowship; H.L. Snyder Medical=20 Foundation; Neidorf Family Brain Tumor Foundation; Stop Cancer = Foundation;=20 Jeanne Schnitzer-Reynolds Fund for Women's Genetic Health; Rosalinde = and=20 Arthur Gilbert Foundation; and Ziman Family Foundation.

  • The costs of publication of this article were defrayed in = part by=20 the payment of page charges. This article must therefore be hereby = marked=20 advertisement in accordance with 18 U.S.C. Section 1734 = solely to=20 indicate this fact.

  • Note: Supplementary data for this = article are=20 available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). =

    • Accepted September = 8, 2008.=20
    • Received March 18, = 2008.=20
    • Revision received = August 18,=20 2008.
  • American = Association=20 for Cancer Research
Previous=20 Section

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    2. The=20 MDM2-p53 Interaction=20
    3. MDM2,=20 An Introduction=20
    4. p63=20 and p73: Roles in Development and Tumor Formation=20
    5. Tamoxifen=20 Functions As a Molecular Agonist Inducing Cell Cycle-Associated = Genes in=20 Breast Cancer Cells
    =C2=BB View = all Most Cited=20 articles

Copyright = =C2=A9 2010=20 American Association for Cancer Research

  • Online ISSN: 1557-3125=20
  • Print ISSN: 1541-7786=20
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normal } #col-2 .toc-navigation .toc-links LI { PADDING-RIGHT: 0px; BACKGROUND-POSITION: 0pt center; DISPLAY: block; = PADDING-LEFT: 9px; FONT-SIZE: 0.85em; BACKGROUND-IMAGE: = url(../../shared/img/misc/rightchevron.gif); PADDING-BOTTOM: 0px; = MARGIN-LEFT: 0px; PADDING-TOP: 2px; BACKGROUND-REPEAT: no-repeat; = WHITE-SPACE: normal } #col-2 .toc-navigation .data-supp-links LI { PADDING-RIGHT: 0px; BACKGROUND-POSITION: 0pt center; DISPLAY: block; = PADDING-LEFT: 9px; FONT-SIZE: 0.85em; BACKGROUND-IMAGE: = url(../../shared/img/misc/rightchevron.gif); PADDING-BOTTOM: 0px; = MARGIN-LEFT: 0px; PADDING-TOP: 2px; BACKGROUND-REPEAT: no-repeat; = WHITE-SPACE: normal } #col-2 .this-issue .toc-links LI { PADDING-RIGHT: 0px; BACKGROUND-POSITION: 0pt center; DISPLAY: block; = PADDING-LEFT: 9px; FONT-SIZE: 0.85em; BACKGROUND-IMAGE: = url(../../shared/img/misc/rightchevron.gif); PADDING-BOTTOM: 0px; = MARGIN-LEFT: 0px; PADDING-TOP: 2px; BACKGROUND-REPEAT: no-repeat; = WHITE-SPACE: normal } #col-2 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PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } SPAN { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } APPLET { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } OBJECT { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } IFRAME { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } H1 { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } H2 { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } H3 { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } H4 { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } H5 { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } H6 { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } P { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } BLOCKQUOTE { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } PRE { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } A { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } ABBR { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } ACRONYM { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } ADDRESS { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } BIG { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } CITE { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } CODE { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } DEL { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } DFN { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } EM { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } FONT { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } IMG { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } INS { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } KBD { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } Q { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } S { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } SAMP { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } SMALL { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } STRIKE { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } STRONG { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } SUB { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } SUP { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } TT { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } VAR { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } DL { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } DT { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } DD { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } OL { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } UL { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } LI { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } FIELDSET { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } FORM { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } LABEL { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } LEGEND { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } TABLE { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } CAPTION { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } TBODY { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } TFOOT { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } THEAD { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } TR { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } TH { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } TD { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; VERTICAL-ALIGN: baseline; PADDING-TOP: 0px; FONT-FAMILY: = inherit; BORDER-RIGHT-WIDTH: 0px; outline-style: none } A IMG { BORDER-TOP-WIDTH: 0px; BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: = 0px; BORDER-RIGHT-WIDTH: 0px } :link IMG { BORDER-TOP-WIDTH: 0px; BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: = 0px; BORDER-RIGHT-WIDTH: 0px } :visited IMG { BORDER-TOP-WIDTH: 0px; BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: = 0px; BORDER-RIGHT-WIDTH: 0px } :focus { outline: none } BODY { BACKGROUND: white; COLOR: black; LINE-HEIGHT: 1.25 } TABLE { BORDER-COLLAPSE: collapse; border-spacing: 0px } CAPTION { FONT-WEIGHT: normal; TEXT-ALIGN: left } TH { FONT-WEIGHT: normal; TEXT-ALIGN: left } TD { FONT-WEIGHT: normal; TEXT-ALIGN: left } BLOCKQUOTE:unknown { content: "" } BLOCKQUOTE:unknown { content: "" } Q:unknown { content: "" } Q:unknown { content: "" } BLOCKQUOTE { quotes: "" "" } Q { quotes: "" "" } BODY { FONT-SIZE: 100%; BACKGROUND: white fixed no-repeat; COLOR: black; = LINE-HEIGHT: 1; FONT-FAMILY: "Lucida Grande", "Lucida Sans Unicode", = Tahoma, Verdana, Arial, Helvetica, sans-serif } P { MARGIN: 1em 5% } PRE { MARGIN: 1em 5% } LABEL { MARGIN: 1em 5% } UL { MARGIN: 1em 5% } OL { MARGIN: 1em 5% } DL { MARGIN: 1em 5% } FIELDSET { MARGIN: 1em 5% } ADDRESS { MARGIN: 1em 5% } H1 { MARGIN: 0px 0px 0.4em } H2 { MARGIN: 0px 0px 0.4em } H3 { MARGIN: 0px 0px 0.4em } H4 { MARGIN: 0px 0px 0.4em } H5 { MARGIN: 0px 0px 0.4em } H6 { MARGIN: 0px 0px 0.4em } H1 { FONT-WEIGHT: bold; TEXT-ALIGN: left } H2 { FONT-WEIGHT: bold; TEXT-ALIGN: left } H3 { FONT-WEIGHT: bold; TEXT-ALIGN: left } H4 { FONT-WEIGHT: bold; TEXT-ALIGN: left } H1 { FONT-SIZE: 1.8em } H2 { FONT-SIZE: 1.5em } H3 { FONT-SIZE: 1.2em } BLOCKQUOTE { MARGIN: 1em 7% } LI { MARGIN-LEFT: 5% } DD { MARGIN-LEFT: 5% } FIELDSET { PADDING-RIGHT: 0.5em; PADDING-LEFT: 0.5em; PADDING-BOTTOM: 0.5em; = PADDING-TOP: 0.5em } EM { FONT-STYLE: italic } STRONG { FONT-WEIGHT: 700 } CODE { FONT-FAMILY: Courier New, Courier, monospace } VAR { FONT-FAMILY: Courier New, Courier, monospace } KBD { FONT-FAMILY: Courier New, Courier, monospace } SAMP { FONT-FAMILY: Courier New, Courier, monospace } PRE { FONT-FAMILY: Courier New, Courier, monospace } SUP { FONT-SIZE: 0.85em; VERTICAL-ALIGN: super; LINE-HEIGHT: 0 } SUB { FONT-SIZE: 0.85em; VERTICAL-ALIGN: sub; LINE-HEIGHT: 0 } DIV.hw-gen-page { PADDING-RIGHT: 4em; PADDING-LEFT: 2em; FONT-SIZE: 0.8em; = PADDING-BOTTOM: 2em; PADDING-TOP: 0.5em } .hw-gen-page #skip-link { LEFT: -9999px; POSITION: absolute } #content-block { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 0px; OVERFLOW: hidden; LINE-HEIGHT: 1.3; PADDING-TOP: 0px; = BORDER-RIGHT-WIDTH: 0px } #content-block P { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = MARGIN: 15px 0px; LINE-HEIGHT: 1.5; PADDING-TOP: 0px; = BORDER-RIGHT-WIDTH: 0px } #content-block { PADDING-RIGHT: 20px; PADDING-LEFT: 5px; FLOAT: right; PADDING-BOTTOM: = 0px; WIDTH: 518px; PADDING-TOP: 0px } #col-2 { OVERFLOW: hidden; WIDTH: 210px } #col-3 { OVERFLOW: hidden; WIDTH: 210px } #col-2 { FLOAT: left } #proxied-contents #col-2 { FLOAT: left } #proxied-contents #col-main { PADDING-RIGHT: 10px; FLOAT: left; OVERFLOW: hidden; WIDTH: 533px } #footer { CLEAR: both } .banner-ads UL { LIST-STYLE-TYPE: none } DIV.hw-gen-page IMG.adborder0 { BORDER-RIGHT: black 0px solid; BORDER-TOP: black 0px solid; = BORDER-LEFT: black 0px solid; BORDER-BOTTOM: black 0px solid } DIV.hw-gen-page IMG.adborder1 { BORDER-RIGHT: black 1px solid; BORDER-TOP: black 1px solid; = BORDER-LEFT: black 1px solid; BORDER-BOTTOM: black 1px solid } DIV.hw-gen-page IMG.adborder2 { BORDER-RIGHT: black 2px solid; BORDER-TOP: black 2px solid; = BORDER-LEFT: black 2px solid; BORDER-BOTTOM: black 2px solid } DIV.hw-gen-page IMG.adborder3 { BORDER-RIGHT: black 3px solid; BORDER-TOP: black 3px solid; = BORDER-LEFT: 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#col-3 A { COLOR: #404040 } #col-3 A:link { COLOR: #404040 } #col-2 A:visited { COLOR: #581858 } #col-3 A:visited { COLOR: #581858 } #col-2 A:hover { COLOR: #404040; BORDER-BOTTOM: #404040 1px dotted; TEXT-DECORATION: = none } #col-3 A:hover { COLOR: #404040; BORDER-BOTTOM: #404040 1px dotted; TEXT-DECORATION: = none } #col-2 A:active { COLOR: #404040 } #col-3 A:active { COLOR: #404040 } #col-3 UL.cover-announce A { COLOR: #2f5085 } #col-3 UL.cover-announce A:link { COLOR: #2f5085 } #col-3 UL.cover-announce A:visited { COLOR: #2f5085 } #col-3 UL.cover-announce A:hover { COLOR: #2f5085; BORDER-BOTTOM: #2f5085 1px dotted; TEXT-DECORATION: = none } #col-3 UL.cover-announce A:active { COLOR: #2f5085 } DIV.sidebar A:hover { TEXT-DECORATION: none } DIV#header { BACKGROUND-POSITION: right top; MARGIN-TOP: 0px; BACKGROUND-IMAGE: = url(/shared/img/standard-design/design2/corner.gif); WIDTH: 960px; = BACKGROUND-REPEAT: no-repeat; BORDER-BOTTOM-STYLE: none } #header #main-logo { MARGIN: 15px 0px 0px 24px; BORDER-TOP-STYLE: none; BORDER-RIGHT-STYLE: = none; BORDER-LEFT-STYLE: none; BORDER-BOTTOM-STYLE: none } #header UL.home-page-ads { PADDING-RIGHT: 10px; PADDING-LEFT: 10px; LEFT: 450px; PADDING-BOTTOM: = 5px; MARGIN: 1.5em 0px 35px; WIDTH: 250px; PADDING-TOP: 5px; = LIST-STYLE-TYPE: none; POSITION: absolute; TOP: 0px } #header UL.home-page-ads LI { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; WIDTH: 250px; PADDING-TOP: 0px; HEIGHT: 40px; TEXT-ALIGN: center } #header UL.home-page-ads LI IMG { BORDER-RIGHT: #aaaaaa 1px solid; BORDER-TOP: #aaaaaa 1px solid; = DISPLAY: block; BORDER-LEFT: #aaaaaa 1px solid; WIDTH: 250px; = BORDER-BOTTOM: #aaaaaa 1px solid; HEIGHT: 40px; BACKGROUND-COLOR: = #e0e0e0 } #header .banner-ads { PADDING-RIGHT: 10px; PADDING-LEFT: 10px; LEFT: 450px; PADDING-BOTTOM: = 5px; MARGIN: 1.5em 0px 35px; WIDTH: 250px; PADDING-TOP: 5px; = LIST-STYLE-TYPE: none; POSITION: absolute; TOP: 0px } .banner-ads#header UL { LIST-STYLE-TYPE: none } #header .banner-ads LI { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; PADDING-TOP: 0px; TEXT-ALIGN: center } #header .banner-ads LI IMG { BORDER-RIGHT: #aaaaaa 1px solid; BORDER-TOP: #aaaaaa 1px solid; = DISPLAY: block; BORDER-LEFT: #aaaaaa 1px solid; BORDER-BOTTOM: #aaaaaa = 1px solid; BACKGROUND-COLOR: #e0e0e0 } #header .header-buttons { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: 5px = 0px 8px 24px; PADDING-TOP: 0px; LIST-STYLE-TYPE: none } #header .header-buttons { COLOR: #0083a8 } #header .header-buttons A { COLOR: #0083a8 } #header .header-buttons LI { PADDING-RIGHT: 0.4em; DISPLAY: inline; PADDING-LEFT: 0.4em; = PADDING-BOTTOM: 0px; MARGIN: 0px; BORDER-LEFT: #666666 1px solid; = PADDING-TOP: 0px } #header .header-buttons LI SPAN { FONT-WEIGHT: bold; FONT-SIZE: 87.5% } #header .header-buttons LI.first { PADDING-LEFT: 0px; BORDER-LEFT-STYLE: none } #header .corner { RIGHT: 0px; POSITION: absolute; TOP: 0px } #header .inst-branding { BORDER-LEFT: #c8c8c8 1px dashed } #header #hdr-login { BORDER-LEFT: #c8c8c8 1px dashed } #header .header-qs { BORDER-LEFT: #c8c8c8 1px dashed } #header .inst-branding { PADDING-LEFT: 5px; LEFT: 756px; POSITION: absolute; TOP: 0px; HEIGHT: = 30px } #header #authstring { DISPLAY: block; LEFT: 0%; WIDTH: 756px; LINE-HEIGHT: 100%; PADDING-TOP: = 5px; POSITION: absolute; TEXT-ALIGN: center } #header #authstring { COLOR: white } #header #authstring LI { PADDING-RIGHT: 2px; PADDING-LEFT: 2px; FONT-SIZE: 87.5%; = PADDING-BOTTOM: 2px; BORDER-TOP-STYLE: none; PADDING-TOP: 2px; = BORDER-RIGHT-STYLE: none; BORDER-LEFT-STYLE: none; LIST-STYLE-TYPE: = none; BORDER-BOTTOM-STYLE: none } #header #authstring A { COLOR: white } #header #hdr-login { PADDING-RIGHT: 0px; PADDING-LEFT: 10px; LEFT: 756px; PADDING-BOTTOM: = 5px; PADDING-TOP: 0px; POSITION: absolute; TOP: 30px } #header #hdr-login FORM LABEL { FONT-SIZE: 87.5%; WHITE-SPACE: nowrap } #header #hdr-login INPUT { FONT-SIZE: 87.5%; WHITE-SPACE: nowrap } #header #hdr-login { COLOR: #0083a8 } #header #hdr-login LABEL { PADDING-RIGHT: 0px; DISPLAY: block; PADDING-LEFT: 0px; FONT-SIZE: = 87.5%; PADDING-BOTTOM: 2px; MARGIN: 0px; WIDTH: 80px; PADDING-TOP: 5px } #header #hdr-login INPUT { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; FONT-SIZE: 87.5%; = PADDING-BOTTOM: 0px; WIDTH: 110px; PADDING-TOP: 0px; HEIGHT: 15px } #header .header-qs INPUT { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; FONT-SIZE: 87.5%; = PADDING-BOTTOM: 0px; WIDTH: 110px; PADDING-TOP: 0px; HEIGHT: 15px } #header #hdr-login INPUT#hdr-login-signin { PADDING-RIGHT: 0px; PADDING-LEFT: 5px; PADDING-BOTTOM: 0px; = VERTICAL-ALIGN: bottom; WIDTH: auto; PADDING-TOP: 0px; HEIGHT: auto } #header .bar { PADDING-LEFT: 20px; MARGIN-LEFT: -20px; WIDTH: 980px; HEIGHT: 60px } #footer .bar { PADDING-LEFT: 20px; MARGIN-LEFT: -20px; WIDTH: 980px; HEIGHT: 60px } #header .bar { BACKGROUND-COLOR: #0083a8 } #footer .bar { BACKGROUND-COLOR: #0083a8 } #header .bar-inner { WIDTH: 960px; COLOR: white; HEIGHT: 60px } #header .bar-inner { BACKGROUND-COLOR: #00a0cc } #footer .footer-group { BACKGROUND-COLOR: #00a0cc } #header .header-qs { PADDING-RIGHT: 5px; DISPLAY: block; PADDING-LEFT: 10px; Z-INDEX: 1; = LEFT: 756px; PADDING-BOTTOM: 7px; MARGIN: 0px; WIDTH: 195px; = PADDING-TOP: 10px; POSITION: absolute; TOP: 112px; HEIGHT: 40px } #header .header-qs LABEL { LEFT: -9999px; POSITION: absolute } #header #hdr-login-signin-label { LEFT: -9999px; POSITION: absolute } #header #header-qs-search-label { LEFT: -9999px; POSITION: absolute } #header .header-qs A { FONT-SIZE: 87.5%; COLOR: white; LINE-HEIGHT: 1.5; WHITE-SPACE: nowrap } #header DIV.adv-search-link A:hover { COLOR: white; BORDER-BOTTOM: white 1px dotted } #header DIV.adv-search-link A:visited { COLOR: white } #header DIV.adv-search-link A:active { COLOR: white } #header DIV.adv-search-link A:link { COLOR: white } #header .header-qs INPUT#header-qs-search-go { MARGIN-LEFT: 0.5em; VERTICAL-ALIGN: bottom; WIDTH: 27px; HEIGHT: auto } #content-block { PADDING-RIGHT: 20px; PADDING-LEFT: 5px; FLOAT: left; PADDING-BOTTOM: = 0px; WIDTH: 518px; PADDING-TOP: 0px } #col-2 { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; PADDING-TOP: 0px } #col-3 { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; PADDING-TOP: 0px } #col-2 { BORDER-RIGHT: #aaaaaa 1px dashed; PADDING-RIGHT: 13px; FLOAT: left; = MARGIN: 0px; OVERFLOW: hidden; WIDTH: 200px } #content-block { COLOR: #403838; BACKGROUND-COLOR: white } .pagetype-proxied #content-block { BORDER-RIGHT: #aaaaaa 1px dashed; PADDING-RIGHT: 0px; PADDING-LEFT: = 0px; PADDING-BOTTOM: 0px; WIDTH: 756px; PADDING-TOP: 0px } .proxied-column-display#proxied-contents #col-main { PADDING-RIGHT: 10px; FLOAT: left; OVERFLOW: hidden; WIDTH: 518px } .proxied-column-display#proxied-contents #col-2 { BORDER-RIGHT: 0px solid; FLOAT: left } #proxied-contents { PADDING-RIGHT: 10px; PADDING-LEFT: 15px } .proxied-column-display#proxied-contents { PADDING-RIGHT: 0px } .proxied-column-display#proxied-contents .search-nav A { FONT-WEIGHT: normal; COLOR: black } .pagetype-proxied #header .banner-ads UL { MARGIN: 1em 5% } .pagetype-proxied #col-3 .content-box H3 { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; FONT-SIZE: 1.2em; = PADDING-BOTTOM: 0.3em; MARGIN: 0px; PADDING-TOP: 0.3em } #proxied-contents TH { FONT-WEIGHT: bold } #proxied-contents TABLE { LINE-HEIGHT: 110% } #proxied-contents TD { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; PADDING-TOP: 0px } TR { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; PADDING-TOP: 0px } TH { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; PADDING-TOP: 0px } P { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; PADDING-TOP: 0px } OL { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; PADDING-TOP: 0px } UL { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; PADDING-TOP: 0px } #proxied-contents H1 { LINE-HEIGHT: 120%; TEXT-ALIGN: left } H3 { LINE-HEIGHT: 120%; TEXT-ALIGN: left } DL { LINE-HEIGHT: 120%; TEXT-ALIGN: left } DT { LINE-HEIGHT: 120%; TEXT-ALIGN: left } DD { LINE-HEIGHT: 120%; TEXT-ALIGN: left } #proxied-contents .proxied-collections H1 { MARGIN-TOP: 0.5em } #col-3 H1 { LINE-HEIGHT: 100%; TEXT-ALIGN: left } H3 { LINE-HEIGHT: 100%; TEXT-ALIGN: left } DL { LINE-HEIGHT: 100%; TEXT-ALIGN: left } DT { LINE-HEIGHT: 100%; TEXT-ALIGN: left } DD { LINE-HEIGHT: 100%; TEXT-ALIGN: left } #proxied-contents #col-3 P { MARGIN: 1em 5% } PRE { MARGIN: 1em 5% } LABEL { MARGIN: 1em 5% } UL { MARGIN: 1em 5% } OL { MARGIN: 1em 5% } DL { MARGIN: 1em 5% } FIELDSET { MARGIN: 1em 5% } ADDRESS { MARGIN: 1em 5% } #col-2 #cb-art-nav LI { MARGIN-TOP: 0.4em } #col-2 DIV.sidebar-issue-nav { MARGIN: 5px 0px 0px 5px } #col-2 DIV.article-nav { MARGIN: 5px 0px 0px 5px } #col-2 DIV.search-nav { MARGIN: 5px 0px 0px 5px } #col-3 { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; FLOAT: left; PADDING-BOTTOM: = 0px; MARGIN: 0px; OVERFLOW: visible; WIDTH: 203px; PADDING-TOP: 0px; = TEXT-ALIGN: left } #col-3 { BACKGROUND-COLOR: white } #col-3 * { POSITION: static } #col-3 DIV { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; PADDING-TOP: 0px } DIV.content-box A:link { COLOR: #404040; TEXT-DECORATION: none } DIV.content-box A:visited { COLOR: #404040; TEXT-DECORATION: none } DIV.content-box A:hover { COLOR: #404040; TEXT-DECORATION: none } DIV.content-box A:active { COLOR: #404040; TEXT-DECORATION: none } #col-3 UL.pub-links LI A:link { BORDER-BOTTOM-WIDTH: 0px; TEXT-DECORATION: none } #col-3 UL.pub-links LI A:visited { BORDER-BOTTOM-WIDTH: 0px; TEXT-DECORATION: none } #col-3 UL.pub-links LI A:hover { BORDER-BOTTOM-WIDTH: 0px; TEXT-DECORATION: none } #col-3 UL.pub-links LI A:active { BORDER-BOTTOM-WIDTH: 0px; TEXT-DECORATION: none } #col-3 UL.ex-pub-links LI A:link { BORDER-BOTTOM-WIDTH: 0px; TEXT-DECORATION: none } #col-3 UL.ex-pub-links LI A:visited { BORDER-BOTTOM-WIDTH: 0px; TEXT-DECORATION: none } #col-3 UL.ex-pub-links LI A:hover { BORDER-BOTTOM-WIDTH: 0px; TEXT-DECORATION: none } #col-3 UL.ex-pub-links LI A:active { BORDER-BOTTOM-WIDTH: 0px; TEXT-DECORATION: none } #col-3 UL.pub-links { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; LEFT: -7px; PADDING-BOTTOM: 0px; = MARGIN: 10px 0px; OVERFLOW: hidden; PADDING-TOP: 0px; POSITION: relative } #col-3 UL.pub-links { LIST-STYLE-TYPE: none } #col-3 UL.pub-links { COLOR: #0083a8 } #col-3 UL.pub-links LI A { COLOR: #0083a8 } #col-3 UL.pub-links LI { BORDER-RIGHT: #aaaaaa 1px dashed; PADDING-RIGHT: 5px; BORDER-TOP: = #aaaaaa 1px dashed; PADDING-LEFT: 4px; FONT-WEIGHT: bold; FONT-SIZE: = 0.85em; PADDING-BOTTOM: 10px; MARGIN: 0px; BORDER-LEFT: #aaaaaa 1px = dashed; PADDING-TOP: 10px; BORDER-BOTTOM: #aaaaaa 1px; POSITION: static; = BACKGROUND-COLOR: white } #col-3 UL.pub-links LI A { BACKGROUND-POSITION: left top; DISPLAY: block; PADDING-LEFT: 1em; = BACKGROUND-IMAGE: = url(/shared/img/standard-design/design2/jnl-link-bullet.gif); = LINE-HEIGHT: 1.2; BACKGROUND-REPEAT: no-repeat } #col-3 UL.pub-links LI.last { BORDER-BOTTOM: #aaaaaa 1px dashed } #col-3 .sidebar-icon-group { PADDING-RIGHT: 20px; PADDING-LEFT: 20px; PADDING-BOTTOM: 20px; = PADDING-TOP: 20px } #col-3 .sidebar-icon-group A:hover { BORDER-BOTTOM: 0px solid } #col-3 UL.ex-pub-links { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; LEFT: -7px; PADDING-BOTTOM: 0px; = MARGIN: 10px 0px; OVERFLOW: hidden; PADDING-TOP: 0px; POSITION: relative } #col-3 UL.ex-pub-links { LIST-STYLE-TYPE: none } #col-3 UL.ex-pub-links { COLOR: #0083a8 } #col-3 UL.ex-pub-links LI A { COLOR: #0083a8 } #col-3 UL.ex-pub-links LI { BORDER-RIGHT: #aaaaaa 1px dashed; PADDING-RIGHT: 5px; BORDER-TOP: = #aaaaaa 1px dashed; PADDING-LEFT: 4px; FONT-WEIGHT: bold; FONT-SIZE: = 75%; PADDING-BOTTOM: 10px; MARGIN: 0px; BORDER-LEFT: #aaaaaa 1px dashed; = PADDING-TOP: 10px; BORDER-BOTTOM: #aaaaaa 1px; POSITION: static; = BACKGROUND-COLOR: white } #col-3 UL.ex-pub-links LI A { BACKGROUND-POSITION: left top; DISPLAY: block; PADDING-LEFT: 1em; = BACKGROUND-IMAGE: = url(/shared/img/standard-design/design2/jnl-link-bullet.gif); = LINE-HEIGHT: 1.2; BACKGROUND-REPEAT: no-repeat } #col-3 UL.ex-pub-links LI.last { BORDER-BOTTOM: #aaaaaa 1px dashed } #col-3 UL.tower-ads { PADDING-RIGHT: 0em; PADDING-LEFT: 0em; LIST-STYLE-IMAGE: none; = PADDING-BOTTOM: 0em; MARGIN: 0em; PADDING-TOP: 0em; LIST-STYLE-TYPE: = none } #col-3 UL.tower-ads LI IMG { PADDING-RIGHT: 0px; DISPLAY: block; PADDING-LEFT: 0px; PADDING-BOTTOM: = 0px; MARGIN: 0em auto 0.5em; PADDING-TOP: 0px } #col-3 .publisher-icon { =09 } #footer { WIDTH: 960px; TEXT-ALIGN: left } #footer { CLEAR: both; BACKGROUND-POSITION: 756px 0px; BACKGROUND-IMAGE: = url(/shared/img/standard-design/design2/dash.gif); PADDING-BOTTOM: 20px; = BACKGROUND-REPEAT: repeat-y; BACKGROUND-COLOR: white } #footer UL { FONT-SIZE: 80%; LIST-STYLE-TYPE: none } #footer .bar { CLEAR: both; POSITION: static } #footer .footer-group { FONT-WEIGHT: bold; COLOR: white; HEIGHT: 60px } #footer .footer-group P { PADDING-RIGHT: 10px; PADDING-LEFT: 10px; PADDING-BOTTOM: 10px; = PADDING-TOP: 10px } #footer .footer-col-left { BORDER-RIGHT: #c8c8c8 1px dashed; FLOAT: left; WIDTH: 756px } #footer .footer-col-right { FLOAT: left; WIDTH: 203px } #footer .footer-col-right UL { PADDING-RIGHT: 0px; DISPLAY: block; PADDING-LEFT: 0px; PADDING-BOTTOM: = 0px; MARGIN: 0px; PADDING-TOP: 8px; LIST-STYLE-TYPE: none } #footer .footer-col-right LI { PADDING-RIGHT: 10px; DISPLAY: block; PADDING-LEFT: 10px; = PADDING-BOTTOM: 4px; BORDER-TOP-STYLE: none; PADDING-TOP: 4px; = BORDER-RIGHT-STYLE: none; BORDER-LEFT-STYLE: none; TEXT-ALIGN: left; = BORDER-BOTTOM-STYLE: none } #footer .copyright { FONT-SIZE: 80%; MARGIN: 1em auto } #footer A:hover { COLOR: white; BORDER-BOTTOM: white 1px dotted } #skip-link { LEFT: -9999px; POSITION: absolute } #header #hdr-login { HEIGHT: 75px } DIV.hw-gen-page .view-more { BORDER-RIGHT: gray 1px solid; BORDER-TOP: gray 1px solid; BORDER-LEFT: = gray 1px solid; BORDER-BOTTOM: gray 1px solid } #header #hdr-login LABEL#hdr-login-signin-label { DISPLAY: inline } ------=_NextPart_000_0000_01CB1972.1BCFA3F0 Content-Type: text/css; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable Content-Location: http://mcr.aacrjournals.org/content/6/12/home-page.css #pageid-home #content-block DIV.main-block { FLOAT: left; MARGIN-BOTTOM: 10px; WIDTH: 350px } #pageid-home #content-block .main-block LI { MARGIN: 0px } #pageid-home #content-block .main-block UL { MARGIN: 0px } #pageid-home #content-block H2 { FONT-SIZE: 1.2em; FONT-FAMILY: Arial, "Lucida Grande", "Lucida Sans = Unicode", Tahoma, Verdana, Helvetica, sans-serif } #pageid-home #content-block H3 { FONT-SIZE: 1.2em; FONT-FAMILY: Arial, "Lucida Grande", "Lucida Sans = Unicode", Tahoma, Verdana, Helvetica, sans-serif } #pageid-home #content-block H2 { COLOR: #0083a8 } #pageid-home #content-block H3 { COLOR: #0083a8 } #pageid-home #content-block H2 A { COLOR: #0083a8 } #pageid-home #content-block DIV.home-announce A { COLOR: #0083a8 } #content-block DIV.home-announce A:hover { BORDER-TOP-WIDTH: 0px; BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: = 0px; BORDER-RIGHT-WIDTH: 0px; TEXT-DECORATION: underline } #pageid-home #content-block H2.current-issue { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 20px; MARGIN: = 0px; PADDING-TOP: 20px } #pageid-home #content-block A.current-issue-link { BORDER-BOTTOM: #0083a8 1px dotted } #pageid-home #content-block .cover-img-wrap { LEFT: 20px; BACKGROUND-IMAGE: = url(/shared/img/standard-design/design2/cover_shadow.png); = BACKGROUND-REPEAT: no-repeat; POSITION: relative; TOP: -25px } #pageid-home #cover-image { LEFT: 5px; POSITION: relative } #pageid-home #cover-image IMG { PADDING-RIGHT: 5px; PADDING-LEFT: 5px; PADDING-BOTTOM: 5px; = BORDER-TOP-STYLE: none; PADDING-TOP: 5px; 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      • ' + $hdr + '
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      ';=0A= if (hasItems) {=0A= var header =3D $this.find("div.most-header h3");=0A= if (header.length) {=0A= header.after(hdrList);=0A= }=0A= }=0A= mostHdrList =3D $this.find("div.most-header ul");=0A= }=0A= // 3) if none of the items are selected, make the first one be=0A= if (((mostHdrList !=3D null) && mostHdrList.length) && = (!hasSelected)) {=0A= modClass(mostHdrList.find("li:first"), "most-cur-sel", "");=0A= modClass($this.children("ul").children("li:first"), "most-cur-sel", = "");=0A= }=0A= // 4) bind a handler to each heading li a to toggle = class=3D"most-cur-sel" for =0A= // that item and its corresponding item in the list below=0A= if (((mostHdrList !=3D null) && mostHdrList.length)) {=0A= mostHdrList.find("li a").click(=0A= function(e) {=0A= var curA =3D $(this);=0A= toggleMostSelection(e, $this, curA.text());=0A= }=0A= );=0A= }=0A= }=0A= );=0A= if (hadMostBoxes) {=0A= fixColHeights(-1);=0A= }=0A= }=0A= =0A= function toggleMostSelection(e, mostItemDiv, groupName) {=0A= if (mostItemDiv.length) {=0A= var hdrItems =3D mostItemDiv.find("div.most-header ul li");=0A= if (hdrItems.length) {=0A= hdrItems.each(=0A= function() {=0A= var hdrName =3D $(this).text();=0A= var isChoice =3D (hdrName =3D=3D groupName);=0A= if (isChoice) {=0A= modClass($(this), 'most-cur-sel', '');=0A= }=0A= else {=0A= modClass($(this), '', 'most-cur-sel');=0A= }=0A= }=0A= );=0A= }=0A= var listItems =3D mostItemDiv.children("ul").find("li");=0A= if (listItems.length) {=0A= listItems.each(=0A= function() {=0A= var itemName =3D $(this).children("h4").text();=0A= var isChoice =3D (itemName =3D=3D groupName);=0A= if (isChoice) {=0A= modClass($(this), 'most-cur-sel', '');=0A= }=0A= else {=0A= modClass($(this), '', 'most-cur-sel');=0A= }=0A= }=0A= );=0A= }=0A= }=0A= fixColHeights(-1);=0A= e.preventDefault();=0A= }=0A= =0A= function toggleCollapse(collapsibleEl, hideTagExpr, modTextTagExpr, = expandStr, contractStr) {=0A= if (typeof(expandStr) =3D=3D "undefined") {=0A= expandStr =3D getSiteOption('expandString','+');=0A= }=0A= if (typeof(contractStr) =3D=3D "undefined") {=0A= contractStr =3D getSiteOption('contractString','-');=0A= }=0A= =0A= var $this =3D collapsibleEl;=0A= if ($this.hasClass('collapsed')) {=0A= modClass($this, 'collapsible', 'collapsed');=0A= }=0A= else if ($this.hasClass('collapsible')) {=0A= modClass($this, 'collapsed', 'collapsible');=0A= }=0A= var $toToggle =3D $this.find(hideTagExpr);=0A= if ($toToggle.length) {=0A= $toToggle.toggle();=0A= }=0A= var $rewrite =3D $this.find(modTextTagExpr);=0A= if ($rewrite.length) {=0A= $rewrite.each(=0A= function(i, e) {=0A= var txt =3D $(e).text();=0A= if (txt =3D=3D expandStr) {=0A= $(e).empty().append(contractStr);=0A= }=0A= else if (txt =3D=3D contractStr) {=0A= $(e).empty().append(expandStr);=0A= }=0A= }=0A= );=0A= }=0A= }=0A= =0A= function setupDockBlock(col, dockId, dockClass, contentRules) {=0A= if ((col =3D=3D 2) || (col =3D=3D 3)) {=0A= var navOrigHeight =3D ((col =3D=3D 2) ? gColOrigHeights.col2 : = gColOrigHeights.col3);=0A= var gColNum =3D col;=0A= var gNav =3D ((col =3D=3D 2) ? gCol2 : gCol3);=0A= var gNavHeight =3D ((gNav.length) ? navOrigHeight : 0);=0A= var gNavTopOffset =3D (gNavHeight > 0 ? gNav.offset().top : 0);=0A= var gScrollPos =3D 0;=0A= var gNavDocked =3D false;=0A= /* this won't work in IE 6, so skip it */=0A= /* article navigation docking block */=0A= $(window).scroll(function () {=0A= if (gNavHeight > 0 && !isIE6) {=0A= var offsets =3D getPageOffset();=0A= var origHeight =3D ((gColNum =3D=3D 2) ? gColOrigHeights.col2 : = gColOrigHeights.col3);=0A= var navBottom =3D (gNavTopOffset + origHeight) - offsets.y;=0A= if (gScrollPos !=3D offsets.y) {=0A= gScrollPos =3D offsets.y;=0A= if ((navBottom <=3D 0) && !gNavDocked) {=0A= /* dock the nav */=0A= gNavDocked =3D true;=0A= addDockBlock(col, dockId, dockClass, contentRules);=0A= }=0A= else if (gNavDocked && (navBottom > 0)) {=0A= /* undock the nav */=0A= gNavDocked =3D false;=0A= removeDockBlock(col, dockId);=0A= }=0A= }=0A= }=0A= });=0A= }=0A= }=0A= function addDockBlock(col, dockId, dockClass, contentRules) {=0A= // get direct children li elements only=0A= var colId =3D ("col-" + col);=0A= var newDiv =3D ' 1) {=0A= for (var i =3D 0; i < (contentRules.length - 1); i =3D i + 2) {=0A= var addTo =3D processDockingRule(contentRules[i]);=0A= var add =3D processDockingRule(contentRules[i + 1]);=0A= var addTarget, addVal;=0A= if (addTo.ruleType =3D=3D 'empty') {=0A= addTarget =3D newDivJQuery;=0A= }=0A= else if (addTo.ruleType =3D=3D 'jQuery') {=0A= addTarget =3D $(addTo.rule);=0A= }=0A= if (add.ruleType =3D 'jQuery') {=0A= var addJQEl =3D $(add.rule);=0A= if (addJQEl.length) {=0A= addVal =3D addJQEl.clone();=0A= }=0A= else {=0A= addVal =3D '';=0A= }=0A= }=0A= else if (add.ruleType =3D 'html') {=0A= addVal =3D add.rule;=0A= }=0A= if (addTarget.length && !(addVal =3D=3D '')) {=0A= addTarget.append(addVal);=0A= }=0A= else {=0A= }=0A= }=0A= }=0A= var newDockJQrule =3D "#" + colId + " #" + dockId;=0A= var newDock =3D $(newDockJQrule);=0A= if (newDock.length) {=0A= prepCollapsibles(newDockJQrule + " div.collapsible, " + newDockJQrule = + " div.collapsed", false);=0A= newDock.fadeIn(250);=0A= }=0A= }=0A= =0A= function processDockingRule(rule) {=0A= var retObj =3D new Object();=0A= retObj.empty =3D (((rule !=3D undefined) && (!(rule =3D=3D ''))) ? = false : true);=0A= if (rule =3D=3D '') {=0A= retObj.ruleType =3D 'empty';=0A= }=0A= else if (/^\$\(.+\)$/.test(rule)) {=0A= retObj.ruleType =3D 'jQuery';=0A= retObj.rule =3D rule.replace(/^\$\((.+)\)$/,"$1");=0A= }=0A= else {=0A= retObj.ruleType =3D 'html';=0A= retObj.rule =3D rule;=0A= }=0A= return retObj;=0A= }=0A= =0A= function removeDockBlock(col, dockId) {=0A= var dockedNav =3D $("div#" + dockId);=0A= if(dockedNav.length) {=0A= dockedNav.fadeOut(250, function() { dockedNav.remove(); });=0A= }=0A= }=0A= =0A= =0A= function updateFormInput(labelMatchString, inputMatchString, = defaultColorString, textColorString) {=0A= if ((defaultColorString =3D=3D null) || (defaultColorString =3D=3D '')) = {=0A= defaultColorString =3D "#A0A0A0";=0A= }=0A= if ((textColorString =3D=3D null) || (textColorString =3D=3D '')) {=0A= textColorString =3D "black";=0A= }=0A= var label =3D $(labelMatchString);=0A= var input =3D $(inputMatchString);=0A= if (input.length) {=0A= if ((label.length) && (input.val() =3D=3D '')) {=0A= input.val(label.text()).css("color",defaultColorString);=0A= }=0A= input.focus(=0A= function(e) {=0A= if ((label.length) && ($(this).val() =3D=3D label.text())) {=0A= $(this).val('').css("color",textColorString);=0A= }=0A= }=0A= );=0A= input.blur(=0A= function(e) {=0A= if ((label.length) && ($(this).val() =3D=3D '')) {=0A= $(this).val(label.text()).css("color",defaultColorString);=0A= }=0A= }=0A= );=0A= }=0A= var parentForm =3D label.parents("form").eq(0);=0A= if (parentForm.length) {=0A= parentForm.submit(=0A= function() {=0A= if ((label.length) && (input.length) && (input.val() =3D=3D = label.text())) {=0A= input.val('');=0A= }=0A= return true;=0A= }=0A= );=0A= }=0A= }=0A= =0A= function checkUnloadedImgs(lookupRule) {=0A= if (lookupRule =3D=3D null) {=0A= lookupRule =3D "img";=0A= }=0A= var imageNum =3D -1;=0A= var images =3D $(lookupRule);=0A= if (images.length) {=0A= imageNum =3D 0;=0A= images.each(=0A= function() {=0A= //if ($(this).height() =3D=3D 0) {=0A= if (this.offsetHeight =3D=3D 0) {=0A= imageNum++;=0A= }=0A= }=0A= );=0A= }=0A= return imageNum;=0A= }=0A= =0A= function fixHeightForImages(iter, numImagesToLoad, lookupRule) {=0A= if (lookupRule =3D=3D null) {=0A= lookupRule =3D "img";=0A= }=0A= if (numImagesToLoad > 0) {=0A= var lastNumImagesToLoad =3D numImagesToLoad;=0A= var newNumImagesToLoad =3D checkUnloadedImgs(lookupRule);=0A= if (newNumImagesToLoad < lastNumImagesToLoad) {=0A= fixColHeights(1);=0A= if ((newNumImagesToLoad > 0) && (iter < 10)) {=0A= setTimeout("fixHeightForImages(" + (iter+1) + "," + = newNumImagesToLoad + ",'" + lookupRule + "')", 1000);=0A= }=0A= }=0A= }=0A= }=0A= function getColInfo(colJQEl) {=0A= var colInfo =3D { valid: false, height: 0, bottom: 0, lastElBottom: 0, = extra: 0, fix: false };=0A= if (colJQEl.length) {=0A= var c =3D colJQEl.children();=0A= if (c.length) {=0A= colInfo.height =3D getObjHeight(colJQEl);=0A= colInfo.bottom =3D colJQEl.offset().top + colInfo.height;=0A= var last =3D c.eq(c.length - 1);=0A= colInfo.lastElBottom =3D last.offset().top + getObjHeight(last);=0A= colInfo.extra =3D colInfo.bottom - colInfo.lastElBottom;=0A= colInfo.valid =3D true;=0A= }=0A= }=0A= return colInfo;=0A= }=0A= function checkColHeights() {=0A= var colToFix =3D 0;=0A= if ((gIsMultiCol || gColTempResize) && ((gColOrigHeights.main + = gColOrigHeights.col2 + gColOrigHeights.col3) > 0)) {=0A= var colMainInfo =3D getColInfo(gColMain);=0A= var col2Info =3D getColInfo(gCol2);=0A= var col3Info =3D getColInfo(gCol3);=0A= if (colMainInfo.valid && (gColLows.main =3D=3D 0)) { gColLows.main =3D = colMainInfo.lastElBottom; }=0A= if (col2Info.valid && (gColLows.col2 =3D=3D 0)) { gColLows.col2 =3D = col2Info.lastElBottom; }=0A= if (col3Info.valid && (gColLows.col3 =3D=3D 0)) { gColLows.col3 =3D = col3Info.lastElBottom; }=0A= // check if low point in any column has changed=0A= if ((colMainInfo.valid) && (colMainInfo.lastElBottom !=3D = gColLows.main)) {=0A= colMainInfo.fix =3D true;=0A= }=0A= if ((col2Info.valid) && (col2Info.lastElBottom !=3D gColLows.col2)) {=0A= col2Info.fix =3D true;=0A= }=0A= if ((col3Info.valid) && (col3Info.lastElBottom !=3D gColLows.col3)) {=0A= col3Info.fix =3D true;=0A= }=0A= // check for columns not having same bottom points=0A= if ((colMainInfo.valid) &&=0A= (((col2Info.valid) && (colMainInfo.height < col2Info.height)) ||=0A= ((col3Info.valid) && (colMainInfo.height < col3Info.height)))) {=0A= colMainInfo.fix =3D true;=0A= }=0A= if ((col2Info.valid) &&=0A= (((colMainInfo.valid) && (col2Info.height < colMainInfo.height)) ||=0A= ((col3Info.valid) && (col2Info.height < col3Info.height)))) {=0A= col2Info.fix =3D true;=0A= }=0A= if ((col3Info.valid) &&=0A= (((colMainInfo.valid) && (col3Info.height < colMainInfo.height)) ||=0A= ((col2Info.valid) && (col3Info.height < col2Info.height)))) {=0A= col3Info.fix =3D true;=0A= }=0A= =0A= if (colToFix > -1) {=0A= if (colMainInfo.valid && colMainInfo.fix) {=0A= if ((col2Info.valid && col2Info.fix) || (col3Info.valid && = col3Info.fix)) {=0A= colToFix =3D -1;=0A= }=0A= else { colToFix =3D 1; }=0A= }=0A= else if (col2Info.valid && col2Info.fix) {=0A= if (col3Info.valid && col3Info.fix) {=0A= colToFix =3D -1;=0A= }=0A= else { colToFix =3D 2; }=0A= }=0A= else if (col3Info.valid && col3Info.fix) {=0A= colToFix =3D 3;=0A= }=0A= }=0A= =0A= if (colToFix !=3D 0) {=0A= fixColHeights(colToFix);=0A= }=0A= if (colMainInfo.valid) { gColLows.main =3D colMainInfo.lastElBottom; }=0A= if (col2Info.valid) { gColLows.col2 =3D col2Info.lastElBottom; }=0A= if (col3Info.valid) { gColLows.col3 =3D col3Info.lastElBottom; }=0A= }=0A= setTimeout("checkColHeights()", 5000);=0A= }=0A= function fixColHeights(colChanged) {=0A= if ((gIsMultiCol || gColTempResize) && getSiteOption('setColSizes', = true)) {=0A= var setAll =3D false;=0A= if (colChanged =3D=3D 1) {=0A= if (gColMain.length) {=0A= setObjHeight(gColMain,'auto');=0A= gColOrigHeights.main =3D getObjHeight(gColMain);=0A= }=0A= }=0A= else if (colChanged =3D=3D 2) {=0A= if (gCol2.length) {=0A= setObjHeight(gCol2,'auto');=0A= gColOrigHeights.col2 =3D getObjHeight(gCol2);=0A= }=0A= }=0A= else if (colChanged =3D=3D 3) {=0A= if (gCol3.length) {=0A= setObjHeight(gCol3,'auto');=0A= gColOrigHeights.col3 =3D getObjHeight(gCol3);=0A= }=0A= }=0A= else {=0A= if (colChanged =3D=3D -1) {=0A= // force resize all 3=0A= if (gColMain.length) { setObjHeight(gColMain,'auto'); }=0A= if (gCol2.length) { setObjHeight(gCol2,'auto'); }=0A= if (gCol3.length) { setObjHeight(gCol3,'auto'); }=0A= }=0A= gColOrigHeights.main =3D ((gColMain.length) ? = (getObjHeight(gColMain)) : 0);=0A= gColOrigHeights.col2 =3D ((gCol2.length) ? (getObjHeight(gCol2)) : 0);=0A= gColOrigHeights.col3 =3D ((gCol3.length) ? (getObjHeight(gCol3)) : 0);=0A= setAll =3D true;=0A= }=0A= if ((gColOrigHeights.main + gColOrigHeights.col2 + = gColOrigHeights.col3) > 0) {=0A= var colInfo;=0A= var maxH =3D Math.max(Math.max(gColOrigHeights.main, = gColOrigHeights.col2), gColOrigHeights.col3);=0A= if ((gColMain.length) && ((gColOrigHeights.main < maxH) || = (getObjHeight(gColMain) > maxH) || setAll)) {=0A= setObjHeight(gColMain, maxH);=0A= colInfo =3D getColInfo(gColMain);=0A= if (colInfo.valid) { gColLows.main =3D colInfo.lastElBottom; }=0A= }=0A= if ((gCol2.length) && ((gColOrigHeights.col2 < maxH) || = (getObjHeight(gCol2) > maxH) || setAll)) {=0A= setObjHeight(gCol2, maxH);=0A= colInfo =3D getColInfo(gCol2);=0A= if (colInfo.valid) { gColLows.col2 =3D colInfo.lastElBottom; }=0A= }=0A= if ((gCol3.length) && ((gColOrigHeights.col3 < maxH) || = (getObjHeight(gCol3) > maxH) || setAll)) {=0A= setObjHeight(gCol3, maxH);=0A= colInfo =3D getColInfo(gCol3);=0A= if (colInfo.valid) { gColLows.col3 =3D colInfo.lastElBottom; }=0A= }=0A= }=0A= }=0A= }=0A= =0A= function getObjHeight(obj) {=0A= if (obj instanceof jQuery) {=0A= return obj.get(0).offsetHeight;=0A= }=0A= else if (obj instanceof Array) {=0A= return obj[0].offsetHeight;=0A= }=0A= else {=0A= return obj.offsetHeight;=0A= }=0A= }=0A= function getObjWidth(obj) {=0A= if (obj instanceof jQuery) {=0A= return obj.get(0).offsetWidth;=0A= }=0A= else if (obj instanceof Array) {=0A= return obj[0].offsetWidth;=0A= }=0A= else {=0A= return obj.offsetWidth;=0A= }=0A= }=0A= function setObjHeight(obj, h) {=0A= if (typeof(h) =3D=3D 'number') {=0A= h =3D "" + h + "px";=0A= }=0A= if (obj instanceof jQuery) {=0A= obj.get(0).style.height =3D h;=0A= }=0A= else if (obj instanceof Array) {=0A= obj[0].style.height =3D h;=0A= }=0A= else {=0A= obj.style.height =3D h;=0A= }=0A= }=0A= function setObjWidth(obj, w) {=0A= if (typeof(w) =3D=3D 'number') {=0A= w =3D "" + w + "px";=0A= }=0A= if (obj instanceof jQuery) {=0A= obj.get(0).style.width =3D w;=0A= }=0A= else if (obj instanceof Array) {=0A= obj[0].style.width =3D w;=0A= }=0A= else {=0A= obj.style.width =3D w;=0A= }=0A= }=0A= =0A= /* num pixels top of page has been scrolled offscreen */=0A= function getPageOffset() {=0A= var offset =3D new Object();=0A= if( typeof( window.pageXOffset ) =3D=3D 'number' ) {=0A= offset.x =3D window.pageXOffset;=0A= offset.y =3D window.pageYOffset;=0A= } else if( document.body && ( document.body.scrollTop ) ) {=0A= offset.x =3D document.body.scrollLeft;=0A= offset.y =3D document.body.scrollTop;=0A= } else if( document.documentElement && ( = document.documentElement.scrollTop ) ) {=0A= offset.x =3D document.documentElement.scrollLeft;=0A= offset.y =3D document.documentElement.scrollTop;=0A= }=0A= if (typeof(offset.x)=3D=3D"undefined") { offset.x =3D 0; }=0A= if (typeof(offset.y)=3D=3D"undefined") { offset.y =3D 0; }=0A= return offset;=0A= }=0A= function getViewportDim() {=0A= var dim =3D new Object();=0A= // non-IE=0A= if (typeof window.innerWidth !=3D 'undefined') {=0A= dim.x =3D window.innerWidth;=0A= dim.y =3D window.innerHeight;=0A= }=0A= // IE6 in standards compliant mode=0A= else if (typeof document.documentElement !=3D 'undefined'=0A= && typeof document.documentElement.clientWidth !=3D=0A= 'undefined' && document.documentElement.clientWidth !=3D 0) {=0A= dim.x =3D document.documentElement.clientWidth;=0A= dim.y =3D document.documentElement.clientHeight;=0A= }=0A= // older versions of IE=0A= else {=0A= dim.x =3D document.getElementsByTagName('body')[0].clientWidth;=0A= dim.y =3D document.getElementsByTagName('body')[0].clientHeight;=0A= }=0A= return dim;=0A= }=0A= =0A= function newWindowTargets() {=0A= var newWins =3D $("a.in-nw");=0A= if (newWins.length) {=0A= newWins.each(=0A= function() {=0A= var $this =3D $(this);=0A= $this.attr("target", "_blank");=0A= modClass($this, 'in-nw-vis', 'in-nw');=0A= }=0A= );=0A= }=0A= }=0A= =0A= function getSiteOption(optionName, defaultVal) {=0A= if (typeof(defaultVal) =3D=3D "undefined") {=0A= defaultVal =3D gSiteOptionUnknown;=0A= }=0A= if (gSiteOptions =3D=3D undefined) {=0A= return defaultVal;=0A= }=0A= else if (gSiteOptions[optionName] =3D=3D undefined) {=0A= return defaultVal;=0A= }=0A= else {=0A= return gSiteOptions[optionName];=0A= }=0A= }=0A= =0A= function addPartHeaders(req) {=0A= req.setRequestHeader('Accept', 'application/xhtml+xml');=0A= req.setRequestHeader('Range', 'part=3Dvariant-contents');=0A= }=0A= function addCommonHeaders(req) {=0A= if (typeof(callbackToken) !=3D 'undefined') {=0A= req.setRequestHeader('X-Token', callbackToken);=0A= }=0A= }=0A= function allowsCookies() {=0A= if (hasTestCookie()) {=0A= return true;=0A= }=0A= else {=0A= setTestCookie();=0A= return hasTestCookie();=0A= }=0A= }=0A= function setTestCookie() {=0A= setCookie('cks','allowed',7,'/',null,null);=0A= }=0A= function hasTestCookie() {=0A= var ckVal =3D getCookie('cks');=0A= if ((ckVal !=3D null) && (ckVal =3D=3D 'allowed')) {=0A= return true;=0A= }=0A= else {=0A= return false;=0A= }=0A= }=0A= =0A= var UIPrefsCk=3D{=0A= name: "UIPrefs",=0A= expDays: 3652,=0A= path: '/'=0A= };=0A= =0A= function prefDefined(name) {=0A= var val =3D getPrefValue(name);=0A= if (val =3D=3D null) {=0A= return false;=0A= }=0A= else {=0A= return true;=0A= }=0A= }=0A= function getPrefValue(name) {=0A= var prefArray =3D getPrefArray();=0A= if (prefArray) {=0A= name =3D convertPrefString(name);=0A= var match =3D name + ":";=0A= for (var i =3D 0; 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'&' : '?') = + 'baseURI=3D' + ((baseAjaxUrl.indexOf('?') > 0) ? = baseAjaxUrl.substring(0, baseAjaxUrl.indexOf('?')): baseAjaxUrl);=0A= //var ajaxUrl =3D baseAjaxUrl + ((href.indexOf('?') >=3D 0) ? '&' : = '?') + 'baseURI=3D' + baseAjaxUrl;=0A= $.ajax({=0A= url: ajaxUrl,=0A= dataType: "html",=0A= type: "GET",=0A= error: ajaxErr,=0A= beforeSend: addFigHeaders,=0A= success: function(xhtml) {=0A= addFig(xhtml, figWrapperEl);=0A= },=0A= complete: ajaxComplete=0A= });=0A= e.preventDefault();=0A= }=0A= function addFigHeaders(req) {=0A= addCommonHeaders(req);=0A= addPartHeaders(req);=0A= }=0A= =0A= function addFig(xhtmlData, figWrapperEl) {=0A= if (xhtmlData && !(xhtmlData.indexOf('=3D 0)) {=0A= figWrapperEl.replaceWith(xhtmlData);=0A= newWindowTargets();=0A= var lookupRule =3D "div.article div.fig img";=0A= var numImagesToLoad =3D checkUnloadedImgs(lookupRule);=0A= setTimeout("fixHeightForImages(1" + "," + numImagesToLoad + ",'" + = lookupRule + "')", 1000);=0A= fixColHeights(1);=0A= }=0A= }=0A= =0A= function refLinksNewWindowTarget() {=0A= $("div.ref-list div.cit-extra a").each(=0A= function(i) {=0A= var origTitle =3D $(this).attr("title");=0A= var newTitle =3D '';=0A= if ((origTitle =3D=3D undefined) || (!origTitle)) {=0A= origTitle =3D '';=0A= }=0A= else {=0A= newTitle =3D origTitle + ' ';=0A= }=0A= newTitle +=3D '[opens in a new window]';=0A= $(this).attr("target", "_blank").attr("title", newTitle);=0A= }=0A= );=0A= }=0A= =0A= function addRefPops() {=0A= var numMissed =3D 0;=0A= var maxToSkip =3D getSiteOption('refPopsMaxToSkip', 10);=0A= var i =3D 1;=0A= var idroot =3D "#xref-ref-";=0A= var el =3D $(idroot + i + '-1');=0A= /* not all refs appear in text; compensate */=0A= while (numMissed < maxToSkip) {=0A= if (el.length) {=0A= numMissed =3D 0;=0A= el.hover(dispRef, hideRef);=0A= var j =3D 2;=0A= var el2 =3D $(idroot + i + "-" + j);=0A= while (el2.length) {=0A= el2.hover(dispRef, hideRef);=0A= j++;=0A= el2 =3D $(idroot + i + "-" + j);=0A= }=0A= }=0A= else {=0A= numMissed++;=0A= }=0A= i++;=0A= el =3D $(idroot + i + "-1");=0A= }=0A= }=0A= =0A= function dispRef(e) {=0A= var link =3D $(this).attr("href");=0A= if($("div#hovering-ref").length) {=0A= $("div#hovering-ref").remove();=0A= //alert("hovering-ref div removed on new hover!");=0A= }=0A= var linkEl =3D $(link);=0A= if (linkEl.length) {=0A= var citHtml =3D linkEl.next("div").children("div.cit-metadata");=0A= if (!(citHtml.length)) {=0A= citHtml =3D linkEl.parent().next("div").children("div.cit-metadata");=0A= }=0A= if (citHtml.length) {=0A= var newDiv =3D '
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        ';=0A= $("body").append(newDiv);=0A= var elH =3D getObjHeight($("div#hovering-ref"));=0A= $("div#hovering-ref").css("left", e.pageX+10).css("top", = e.pageY-elH).css("position", "absolute");=0A= }=0A= }=0A= }=0A= =0A= function hideRef(e) {=0A= if($("div#hovering-ref").length) {=0A= $("div#hovering-ref").remove();=0A= }=0A= }=0A= =0A= function getHWCiting() {=0A= var citingA =3D $("#cb-hw-citing-articles");=0A= if (citingA.length) {=0A= var newA =3D 'Loading citing = article data...';=0A= citingA.replaceWith(newA);=0A= var href =3D citingA.attr("href");=0A= var id =3D '';=0A= if (href && (href.indexOf('?') > 0)) {=0A= var args =3D href.substring(href.indexOf('?') + 1).split('&');=0A= for (var i =3D 0; i < args.length; i++) {=0A= if (args[i].toLowerCase().indexOf('legid=3D') =3D=3D 0) {=0A= id =3D args[i].substring(args[i].indexOf('=3D') + 1);=0A= if (id.indexOf('#') > 0) {=0A= id =3D id.substring(0, id.indexOf('#'));=0A= }=0A= }=0A= }=0A= if (!(id =3D=3D '')) {=0A= var host =3D document.location.protocol + "//" + = document.location.host;=0A= var ajaxUrl =3D host + '/cited-by/' + id.replace(/;/,'/');=0A= $.ajax({=0A= url: ajaxUrl,=0A= dataType: "html",=0A= type: "GET",=0A= error: ajaxErr,=0A= success: addHWCiting,=0A= complete: ajaxComplete=0A= });=0A= =0A= }=0A= }=0A= }=0A= }=0A= function getHWRelatedURLs() {=0A= var relatedURLsA =3D $("#cb-related-urls");=0A= var relatedURLsMsg =3D = getSiteOption('relatedWebPageLoadingText','Loading related web page = data...'); =0A= if (relatedURLsA.length) {=0A= var newA =3D '' + = relatedURLsMsg + '';=0A= relatedURLsA.replaceWith(newA);=0A= var href =3D relatedURLsA.attr("href");=0A= var id =3D '';=0A= if (href && (href.indexOf('?') > 0)) {=0A= var args =3D href.substring(href.indexOf('?') + 1).split('&');=0A= for (var i =3D 0; i < args.length; i++) {=0A= if (args[i].toLowerCase().indexOf('legid=3D') =3D=3D 0) {=0A= id =3D args[i].substring(args[i].indexOf('=3D') + 1);=0A= if (id.indexOf('#') > 0) {=0A= id =3D id.substring(0, id.indexOf('#'));=0A= }=0A= }=0A= }=0A= if (!(id =3D=3D '')) {=0A= var host =3D document.location.protocol + "//" + = document.location.host;=0A= var ajaxUrl =3D host + '/related-web-pages/' + id.replace(/;/,'/');=0A= $.ajax({=0A= url: ajaxUrl,=0A= dataType: "html",=0A= type: "GET",=0A= error: ajaxErr,=0A= success: addRelatedURLs,=0A= complete: ajaxComplete=0A= });=0A= =0A= }=0A= }=0A= }=0A= }=0A= function getPatientInformData() {=0A= var pInformA =3D $("#cb-patientinform");=0A= if (pInformA.length) {=0A= var newA =3D 'Loading = patientINFORMation...';=0A= pInformA.replaceWith(newA);=0A= var href =3D pInformA.attr("href");=0A= var id =3D '';=0A= if (href && (href.indexOf('?') > 0)) {=0A= var args =3D href.substring(href.indexOf('?') + 1).split('&');=0A= for (var i =3D 0; i < args.length; i++) {=0A= if (args[i].toLowerCase().indexOf('legid=3D') =3D=3D 0) {=0A= id =3D args[i].substring(args[i].indexOf('=3D') + 1);=0A= if (id.indexOf('#') > 0) {=0A= id =3D id.substring(0, id.indexOf('#'));=0A= }=0A= }=0A= }=0A= if (!(id =3D=3D '')) {=0A= var host =3D document.location.protocol + "//" + = document.location.host;=0A= var ajaxUrl =3D host + '/related-web-pages/patientinform/' + = id.replace(/;/,'/');=0A= $.ajax({=0A= url: ajaxUrl,=0A= dataType: "html",=0A= type: "GET",=0A= error: ajaxErr,=0A= success: addPatientInform,=0A= complete: ajaxComplete=0A= });=0A= =0A= }=0A= }=0A= }=0A= }=0A= function getISIRelated() {=0A= var relatedA =3D $("#cb-isi-similar-articles");=0A= if (relatedA.length) {=0A= var newA =3D 'Loading Web = of Science article data...';=0A= relatedA.replaceWith(newA);=0A= var href =3D relatedA.attr("href");=0A= var id =3D '';=0A= if (href) {=0A= var hrefDec =3D decodeURI(href);=0A= if ((hrefDec.indexOf('?') > 0)) {=0A= var args =3D hrefDec.substring(hrefDec.indexOf('?') + 1).split('&');=0A= for (var i =3D 0; i < args.length; i++) {=0A= var argDec =3D decodeURIComponent(args[i]);=0A= if (argDec.toLowerCase().indexOf('access_num=3D') =3D=3D 0) {=0A= id =3D argDec.substring(argDec.indexOf('=3D') + 1);=0A= if (id.indexOf('#') > 0) {=0A= id =3D id.substring(0, id.indexOf('#'));=0A= }=0A= }=0A= }=0A= if (!(id =3D=3D '')) {=0A= var host =3D document.location.protocol + "//" + = document.location.host;=0A= var ajaxUrl =3D host + '/isi-links/has-related/' + = id.replace(/;/,'/');=0A= $.ajax({=0A= url: ajaxUrl,=0A= dataType: "html",=0A= type: "GET",=0A= error: ajaxErr,=0A= success: addISIRelated,=0A= complete: ajaxComplete=0A= });=0A= }=0A= }=0A= }=0A= }=0A= }=0A= function getCiting(service, msg, pathseg, successFn) {=0A= var citingA =3D $("#cb-" + service + "-citing-articles");=0A= if (citingA.length) {=0A= var newA =3D '' = + msg + '';=0A= citingA.replaceWith(newA);=0A= var href =3D citingA.attr("href");=0A= var id =3D '';=0A= if (href) {=0A= var hrefDec =3D decodeURI(href);=0A= if ((hrefDec.indexOf('?') > 0)) {=0A= var args =3D hrefDec.substring(hrefDec.indexOf('?') + 1).split('&');=0A= for (var i =3D 0; i < args.length; i++) {=0A= var argDec =3D decodeURIComponent(args[i]);=0A= if (argDec.toLowerCase().indexOf('access_num=3D') =3D=3D 0) {=0A= id =3D argDec.substring(argDec.indexOf('=3D') + 1);=0A= if (id.indexOf('#') > 0) {=0A= id =3D id.substring(0, id.indexOf('#'));=0A= }=0A= }=0A= }=0A= if (!(id =3D=3D '')) {=0A= var host =3D document.location.protocol + "//" + = document.location.host;=0A= var ajaxUrl =3D host + '/' + service + '-links/' + pathseg + = id.replace(/;/,'/');=0A= $.ajax({=0A= url: ajaxUrl,=0A= dataType: "html",=0A= type: "GET",=0A= error: ajaxErr,=0A= success: successFn,=0A= complete: ajaxComplete=0A= });=0A= }=0A= }=0A= }=0A= }=0A= }=0A= =0A= function getISICiting() {=0A= var citingA =3D $("#cb-isi-citing-articles");=0A= if (citingA.length) {=0A= var newA =3D 'Loading Web of = Science citing article data...';=0A= citingA.replaceWith(newA);=0A= var href =3D citingA.attr("href");=0A= var id =3D '';=0A= if (href) {=0A= var hrefDec =3D decodeURI(href);=0A= if ((hrefDec.indexOf('?') > 0)) {=0A= var args =3D hrefDec.substring(hrefDec.indexOf('?') + 1).split('&');=0A= for (var i =3D 0; i < args.length; i++) {=0A= var argDec =3D decodeURIComponent(args[i]);=0A= if (argDec.toLowerCase().indexOf('access_num=3D') =3D=3D 0) {=0A= id =3D argDec.substring(argDec.indexOf('=3D') + 1);=0A= if (id.indexOf('#') > 0) {=0A= id =3D id.substring(0, id.indexOf('#'));=0A= }=0A= }=0A= }=0A= if (!(id =3D=3D '')) {=0A= var host =3D document.location.protocol + "//" + = document.location.host;=0A= var ajaxUrl =3D host + '/isi-links/' + id.replace(/;/,'/');=0A= $.ajax({=0A= url: ajaxUrl,=0A= dataType: "html",=0A= type: "GET",=0A= error: ajaxErr,=0A= success: addISICiting,=0A= complete: ajaxComplete=0A= });=0A= }=0A= }=0A= }=0A= }=0A= }=0A= function getEntrezLinks() {=0A= var entrezDiv =3D $("#cb-entrez-links-placeholder");=0A= if (entrezDiv.length) {=0A= var entrezA =3D entrezDiv.children("a");=0A= if (entrezA) {=0A= var host =3D document.location.protocol + "//" + = document.location.host;=0A= var ajaxUrl =3D host + entrezA.attr("href");=0A= $.ajax({=0A= url: ajaxUrl,=0A= dataType: "html",=0A= type: "GET",=0A= error: ajaxErr,=0A= success: addEntrezLinks,=0A= complete: ajaxComplete=0A= });=0A= }=0A= }=0A= }=0A= =0A= function ajaxErr(req, msg, e) {=0A= }=0A= function ajaxComplete(req, msg) {=0A= }=0A= function updateCBItem(cbItem, newHTML, hasData) {=0A= var parentItem =3D cbItem.parents("li").eq(0);=0A= cbItem.replaceWith(newHTML);=0A= if (!hasData) {=0A= // hide the parent li=0A= if (parentItem.length) {=0A= modClass(parentItem,"nodata","");=0A= // check if there are any siblings still being displayed=0A= var otherItems =3D parentItem.siblings();=0A= var allItemsEmpty;=0A= if (otherItems.length) {=0A= if (otherItems.length =3D=3D otherItems.filter(".nodata").length) {=0A= allItemsEmpty =3D true=0A= }=0A= else {=0A= allItemsEmpty =3D false;=0A= }=0A= }=0A= else {=0A= allItemsEmpty =3D true;=0A= }=0A= if (allItemsEmpty) {=0A= var cbsection =3D parentItem.parents("div.cb-section").eq(0);=0A= if (cbsection.length) {=0A= modClass(cbsection,"nodata","");=0A= }=0A= }=0A= }=0A= }=0A= // in frameset fix targets on child links, forms=0A= fixFrameLinks(parentItem.find("a,form"));=0A= fixColHeights(2);=0A= }=0A= function fixFrameLinks(jqItems) {=0A= if ((gIsFrameset !=3D null) && gIsFrameset) {=0A= jqItems.each(=0A= function(i) {=0A= var href =3D $(this).attr("href");=0A= var action =3D $(this).attr("action"); // if form=0A= if ((href !=3D null) || (action !=3D null)) {=0A= var inFrameAnchor =3D ((href !=3D null) && = (((/frameset=3D/.test(href)) && (/#/.test(href))) || = (href.substring(0,1) =3D=3D '#')));=0A= if ((!inFrameAnchor) || (action !=3D null)) {=0A= $(this).attr("target", "_top");=0A= }=0A= }=0A= }=0A= );=0A= }=0A= }=0A= function addRelatedURLs(xhtmlData) {=0A= var cbA =3D $("#cb-loading-related-urls");=0A= if (gIsFrameset) {=0A= if (cbA.length) {=0A= updateCBItem(cbA, '', false);=0A= }=0A= }=0A= else if (xhtmlData && !(xhtmlData.indexOf('=3D 0)) {=0A= $("#related-urls").replaceWith(xhtmlData);=0A= var relatedWebPagesLabel =3D getSiteOption('relatedWebPagesLabel', = 'Related Web Pages');=0A= fixColHeights(1);=0A= if (cbA.length) {=0A= updateCBItem(cbA, '' + relatedWebPagesLabel = + '', true);=0A= }=0A= }=0A= else {=0A= if (cbA.length) {=0A= updateCBItem(cbA, '', false);=0A= }=0A= }=0A= }=0A= function addPatientInform(xhtmlData) {=0A= var cbA =3D $("#cb-loading-patientinform");=0A= if (gIsFrameset) {=0A= if (cbA.length) {=0A= updateCBItem(cbA, '
        Not = available in this view
        ', false);=0A= }=0A= }=0A= else if (xhtmlData && !(xhtmlData.indexOf('=3D 0)) {=0A= $("#patientinform-links").replaceWith(xhtmlData);=0A= fixColHeights(1);=0A= if (cbA.length) {=0A= updateCBItem(cbA, 'patientINFORMation', true);=0A= }=0A= }=0A= else {=0A= if (cbA.length) {=0A= updateCBItem(cbA, '
        No = patientINFORMation available for this article
        ', false);=0A= }=0A= }=0A= }=0A= function addHWCiting(xhtmlData) {=0A= var cbA =3D $("#cb-loading-hw-cited");=0A= if (gIsFrameset) {=0A= if (cbA.length) {=0A= updateCBItem(cbA, '
        Not available = in this view
        ', false);=0A= }=0A= }=0A= else if (xhtmlData) {=0A= $("#content-block").append(xhtmlData);=0A= var hwCitingLabel =3D getSiteOption('hwCitingLabel', 'View citing = article information');=0A= fixColHeights(1);=0A= if (cbA.length) {=0A= updateCBItem(cbA, '' + hwCitingLabel + '', = true);=0A= }=0A= }=0A= else {=0A= if (cbA.length) {=0A= updateCBItem(cbA, '
        No citing = articles
        ', false);=0A= }=0A= }=0A= }=0A= function addISIRelated(xhtmlData) {=0A= var cbA =3D $("#cb-isi-similar-articles");=0A= if (xhtmlData && !(xhtmlData.indexOf('=3D 0)) {=0A= if (cbA.length) {=0A= updateCBItem(cbA, xhtmlData, true);=0A= }=0A= }=0A= else {=0A= if (cbA.length) {=0A= updateCBItem(cbA, '', false);=0A= }=0A= }=0A= }=0A= function addISICiting(xhtmlData) {=0A= var cbA =3D $("#cb-loading-isi-cited");=0A= if (xhtmlData && !(xhtmlData.indexOf('=3D 0)) {=0A= if (cbA.length) {=0A= updateCBItem(cbA, xhtmlData, true);=0A= }=0A= }=0A= else {=0A= if (cbA.length) {=0A= updateCBItem(cbA, '
        No Web of = Science citing articles
        ', false);=0A= }=0A= }=0A= }=0A= function addScopusCiting(xhtmlData) {=0A= var cbA =3D $("#cb-loading-scopus-cited");=0A= if (xhtmlData && (xhtmlData.indexOf('=3D 0)) {=0A= if (cbA.length) {=0A= updateCBItem(cbA, xhtmlData, true);=0A= }=0A= }=0A= else {=0A= if (cbA.length) {=0A= updateCBItem(cbA, '
        No Scopus = citing articles
        ', false);=0A= }=0A= }=0A= }=0A= function addEntrezLinks(xhtmlData) {=0A= var entrezDiv =3D $("#cb-entrez-links-placeholder");=0A= =0A= if (xhtmlData && (xhtmlData.indexOf('        =3D 0)) {=0A= if (entrezDiv.length) {=0A= updateCBItem(entrezDiv, xhtmlData, true);=0A= }=0A= }=0A= else {=0A= if (entrezDiv.length) {=0A= updateCBItem(entrezDiv, '', false);=0A= }=0A= }=0A= }=0A= =0A= function updateSBLinks() {=0A= var fbLink =3D $("a.sb-facebook");=0A= if (fbLink.length) {=0A= fbLink.click(=0A= function(e) {=0A= window.open(this.href, 'sharer', = 'toolbar=3D0,status=3D0,width=3D626,height=3D436');=0A= e.preventDefault();=0A= }=0A= );=0A= }=0A= }=0A= =0A= function addDockedNav() {=0A= var slugEl =3D $("#col-2 #slugline");=0A= // get direct children li elements only=0A= var artViews =3D $("#article-cb-main .cb-section ol:has(li = a[rel^=3D'view-']) > li").clone();=0A= var newDiv =3D '
        ';=0A= $("#col-2").append(newDiv);=0A= $("#col-2 #docked-nav").hide();=0A= var newDivJQuery =3D $("#docked-nav");=0A= newDivJQuery.append('

        This Article

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