From: Subject: =?Windows-1252?Q?Identification_of_Potential_Serum_Biomarkers_of_Glioblas?= =?Windows-1252?Q?toma:_Serum_Osteopontin_Levels_Correlate_with_Poor_Progn?= =?Windows-1252?Q?osis_=97_Cancer_Epidemiology=2C_Biomarkers_&_Prevention?= Date: Thu, 29 Jul 2010 16:26:31 +0200 MIME-Version: 1.0 Content-Type: multipart/related; type="text/html"; boundary="----=_NextPart_000_0000_01CB2F3A.CD86C000" X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.5931 This is a multi-part message in MIME format. ------=_NextPart_000_0000_01CB2F3A.CD86C000 Content-Type: text/html; charset="utf-8" Content-Transfer-Encoding: quoted-printable Content-Location: http://cebp.aacrjournals.org/content/19/6/1409.long =EF=BB=BF Identification of = Potential Serum Biomarkers of Glioblastoma: Serum Osteopontin Levels = Correlate with Poor Prognosis =E2=80=94 Cancer Epidemiology, Biomarkers = & Prevention
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Identification of Potential Serum Biomarkers of = Glioblastoma: Serum Osteopontin Levels Correlate with Poor = Prognosis

  1. Peddagangannagari = Sreekanthreddy1,= =20
  2. Harish=20 Srinivasan2,= =20
  3. Durairaj=20 Mohan Kumar1,= =20
  4. Mamatha=20 Bangalore Nijaguna1,= =20
  5. Sambandam=20 Sridevi2,= =20
  6. Marigowda=20 Vrinda3,= =20
  7. Arimappamagan=20 Arivazhagan4,= =20
  8. Anandh=20 Balasubramaniam4,= =20
  9. Alangar=20 Sathyaranjandas Hegde5,= =20
  10. Bangalore=20 A. Chandramouli4,= =20
  11. Vani=20 Santosh4,= =20
  12. Manchanahalli=20 R.S. Rao6,= =20
  13. Paturu=20 Kondaiah2 = and=20
  14. Kumaravel=20 Somasundaram1 =
  1. Authors' Affiliations: Departments of=20 1Microbiology and Cell Biology and 2Molecular=20 Reproduction, Development and Genetics, Indian Institute of Science;=20 Departments of 3Neuropathology and = 4Neurosurgery,=20 National Institute of Mental Health and Neuro Sciences; = 5Sri Satya=20 Sai Institute of Higher Medical Sciences; and 6Jawaharlal = Nehru=20 Centre for Advanced Scientific Research, Bangalore, India =
  1. Corresponding = Author:
    Kumaravel=20 Somasundaram, Department of Microbiologyand Cell Biology, Indian = Institute of=20 Science, Bangalore 560012, India. Phone: 91-80-23607171; Fax: = 91-80-23602697.=20 E-mail: skumar{at}mcbl.iisc.ernet.in =
Next=20 Section

Abstract

Background: The aim of this study is to = identify=20 serum biomarkers with classification and prognosis utility for = astrocytoma, in=20 particular glioblastoma (GBM).

Methods: Our previous glioma microarray = database was=20 mined to identify genes that encode secreted or membrane-localized = proteins.=20 Subsequent analysis was done using significant analysis of microarrays, = followed=20 by reverse transcription-quantitative PCR (RT-qPCR) and = immunohistochemical=20 validation in tumor tissues, ELISA and Western blot validation in sera, = and=20 correlation with survival of GBM patients.

Results: Significant analysis of = microarrays=20 identified 31 upregulated and 3 downregulated genes specifically in = GBMs.=20 RT-qPCR validation on an independent set of samples confirmed the = GBM-specific=20 differential expression of several genes, including three upregulated = (CALU,=20 CXCL9, and TIMP1) and two downregulated (GPX3 and = TIMP3) novel genes. With respect to osteopontin (OPN), we show = the=20 GBM-specific upregulation by RT-qPCR and immunohistochemical staining of = tumor=20 tissues. Elevated serum OPN levels in GBM patients were also shown by = ELISA and=20 Western blot. GBM patients with high serum OPN levels had poorer = survival than=20 those with low serum OPN levels (median survival 9 versus 22 months=20 respectively; P =3D 0.0001). Further, we also show high serum = TIMP1=20 levels in GBM patients compared with grade II/III patients by ELISA and=20 downregulation of serum GPX3 and TIMP3 proteins in GBMs compared with = normal=20 control by Western blot analysis.

Conclusions: Several novel potential serum = biomarkers=20 of GBM are identified and validated. High serum OPN level is found as a = poor=20 prognostic indicator in GBMs.

Impact: Identified serum biomarkers may = have=20 potential utility in astrocytoma classification and GBM prognosis. = Cancer=20 Epidemiol Biomarkers Prev; 19(6); 1409=E2=80=9322. =C2=A92010 AACR. =

<= SPAN>Previous=20 Section= Next=20 Section

This article is featured in Highlights of This Issue, p. = 1387

Previous=20 Section= Next=20 Section

Introduction

An immediate need in oncology is biomarkers, which can be = identified=20 through simple noninvasive or less invasive methods with the potential = to=20 identify cancer risk, promote early diagnosis, grade accurately, and = monitor=20 treatment response (1,= 2)= . Serum=20 seems to be a good choice for this purpose because it can be easily = obtained=20 from patients and evaluated for expression of biomarkers. Blood = components are=20 also highly dynamic that it can reflect the normal or abnormal status of = an=20 individual (1)= . Genomic=20 and proteomic approaches using high-throughput techniques have resulted = in=20 identification of several novel genes and their products associated with = cancer.=20 Secreted proteins can be used as circulating tumor markers for diagnosis = and=20 monitoring patient response to treatment.

Gliomas are the most common primary central nervous system = tumors of=20 the brain. They include astrocytomas, oligodendrogliomas, and = oligoastrocytomas=20 based on type of cells they originate from, with the latter having = components of=20 both cell types (3)= .=20 Astrocytomas are further divided into pilocytic astrocytoma (grade I), = diffuse=20 astrocytoma (DA; grade II), anaplastic astrocytoma (AA; grade III), and=20 glioblastoma (GBM; grade IV), whereas oligodendrogliomas are classified = as=20 diffuse oligodendroglioma (DO; grade II) and anaplastic = oligodendroglioma (AO;=20 grade III). Pilocytic tumors are slow growing, noninfiltrating, and = usually=20 cured by surgery. Although malignant gliomas are represented by AA, AO, = and=20 GBMs, patients with GBM have the worst prognosis with a median survival = <12=20 months despite advances in surgery, radiation therapy, and chemotherapy = (4)= . The=20 situation is further complicated by existing histopathology-based=20 classification, as it is likely to be subjective and also suffers from=20 considerable interobserver variation (5)= .=20 Accurate classification seems to be important as the treatment = modalities are=20 substantially different between grade III and IV patients (6,= 7)= .=20 Moreover, the existence of subclass with varying responses to treatment = within=20 similar histologic grades of glioma has been shown (8)= . It is=20 believed that classification based on gene signatures has a good chance = of=20 achieving clinically relevant subgroups. This warrants a need for = identification=20 of more reliable biomarkers with extensive validation.

A number of serum biomarkers with multiple utility for = glioma have=20 been reported (2)= .=20 However, none of these markers are approved for routine use for = patients. With=20 an aim to identify new serum biomarkers that could classify glioma = grades more=20 precisely and provide prognostic value among GBM patients, we carried = out data=20 mining of our glioma microarray database (9)= and=20 identified genes that are located in the membrane or secreted based on = protein=20 localization information obtained from the SOURCE database as potential = serum=20 biomarkers. Subsequent analysis by significant analysis of microarrays = (SAM),=20 followed by reverse transcription-quantitative PCR (RT-qPCR) validation, = identified several new GBM-specific serum markers. For selected genes, = we=20 carried out additional validations. For one gene, that is, = osteopontin=20 (OPN), the validation was also carried out by = immunohistochemical=20 analysis using tissue sections and ELISA using serum samples. Serum OPN = level=20 was also correlated with patient survival. With this, we have identified = several=20 secreted biomarkers, some of which are potential serum markers of GBM.=20

= Previous=20 Section= Next=20 Section

Materials and Methods

Tumor samples

Tumor samples were collected from patients who were = operated at=20 National Institute of Mental Health and Neurosciences (NIMHANS) and Sri = Satya=20 Sai Institute of Higher Medical Sciences (SSIHMS), Bangalore, India. = Normal=20 brain tissue samples (anterior temporal lobe) obtained during surgery = for=20 intractable epilepsy were used as control samples. Informed consent from = each=20 patient was obtained per IEC guidelines and approval. A total of 118 = samples,=20 which included different grades of astrocytomas: 8 DA (grade II), 26 AA = (grade=20 III), 44 GBM (grade IV), oligodendroglioma including 7 DO (grade II) and = 26 AO=20 (grade III), and 7 normal brain samples were used for real-time qPCR = analysis in=20 this study. Tissues were bisected and one-half was snap-frozen in liquid = nitrogen and stored at =E2=88=9280=C2=B0C until RNA isolation. The other = half was fixed in=20 formalin and processed for paraffin sections, and these tissues were = used for=20 histopathologic characterization and immunohistochemistry.

Microarray data and SAM analyses, RNA isolation, and RT-qPCR and = Western=20 blot analyses

The details, as previously described, are given in the = supplementary=20 section (9-= 12).

Histopathology and immunohistochemistry

Histologic sections of normal brain and tumor tissues were = examined=20 by light microscopy using hematoxylin and eosin preparation. Tumor = sections of=20 diffusely infiltrating astrocytomas were graded using the WHO grading = scheme (13). A=20 total of 82 cases (78 cases of diffusely infiltrating astrocytoma and 4 = control=20 brain tissues) were analyzed by immunohistochemistry. Paraffin sections = (4 =CE=BCm)=20 from the tumor tissue and control samples were collected on = silane-coated=20 slides, and immunohistochemistry was done using an antibody against OPN=20 (polyclonal: ab8448, Abcam, UK, diluted to 1:200). Antigen retrieval was = not=20 required for OPN staining. After the initial processing steps, sections = were=20 incubated with the primary antibody, overnight at 4=C2=B0C. This was = followed by=20 incubation with Super Sensitive poly=E2=80=93horseradish peroxidase = detection system=20 (QD440-XAK, Biogenex). 3,3=E2=80=B2-Diaminobenzidine (Sigma) was used as = the chromogenic=20 substrate. The tumor sample, which showed significant upregulation of = OPN by=20 qPCR, served as the positive control. A negative control slide in which = the=20 primary antibody was excluded was incorporated with each batch of = slides.=20 Staining was scored on a scale of 0 to 2 (0, no staining; 1+, mild to = moderate=20 intensity; 2+, strong staining). Only 2+ staining was considered for=20 interpretation.

Serum collection, OPN, and TIMP1 ELISA

Serum collection and storage conditions are described = previously (12). OPN=20 levels were determined in serum samples using the human OPN immunoassay = kit from=20 R&D Systems according to the manufacturer's protocol. Human TIMP1 = ELISA=20 quantitation kit from R&D Systems was used to measure serum TIMP1 = levels per=20 the manufacturer's instructions. More details can be seen in the = supplementary=20 section.

Survival correlation with serum OPN levels

Newly diagnosed GBM patients (n =3D 30) who = underwent surgery=20 in the two clinical centers (NIMHANS/SSIHMS) were prospectively = included.=20 Approval by the ethics committee and patient consent were obtained = before=20 initiation of the study. The patients were recruited based on the = following=20 inclusion criteria: (a) adult patients (age between 18 and 65 = years)=20 with a supratentorial lobar tumor; (b) patients who underwent = maximal=20 safe resection of the tumor with minimal residue noted on postoperative = magnetic=20 resonance imaging scan; and (c) patients with postoperative = Karnofsky's=20 performance score =E2=89=A570. The demographic data and clinical = features of these=20 patients were noted. Blood was collected from all patients = preoperatively, and=20 serum OPN levels were assessed by the methods described above.

All patients were treated uniformly with adjuvant = radiotherapy and=20 chemotherapy. Radiotherapy was administered with a total dose of 59.4 = Gy, given=20 in 33 fractions along with concomitant chemotherapy with temozolamide,=20 administered at the dose of 100 mg/d, which was continued daily for 45 = days.=20 Subsequently, five cycles of cyclical chemotherapy with temozolamide at = the dose=20 of 150 mg/m2 body surface area for 5 days every 28 days was=20 administered.

Patients were followed up clinically and with magnetic = resonance=20 imaging. Re-surgery was offered for those who developed symptomatic = recurrence.=20 The maximum follow-up period was 34 months. At the end of study period, = 23=20 patients had expired and 7 were on follow up. The median survival of = patients in=20 the whole group was 15 months.

= Previous=20 SectionNext=20 Section

Results

Analysis of secreted or plasma membrane=E2=80=93associated genes in = astrocytoma=20 microarray expression profile database

With the idea that genes that encode protein located in the = plasma=20 membrane or secreted are likely to be potential serum biomarkers, we = analyzed=20 our recently published microarray data (9)= . Using=20 protein localization information from the SOURCE database, we identified = 226=20 such genes from a total of 18,981 genes that were spotted in the = microarray. In=20 the next step, we used SAM to find genes that are differentially = expressed=20 between GBM and lower-grade astrocytomas (grade II/III). Of the total of = 34=20 genes that are differentially expressed, 31 were upregulated in GBMs as = against=20 lower-grade astrocytomas (grade II/III) and 3 were downregulated in GBM = as=20 against lower-grade astrocytomas (grade II/III) were identified (Table = 1). To=20 get a visual appreciation as well as to see the substructure of the data = containing expression values of 34 genes, we carried out hierarchical = cluster=20 analysis using the TMEV software (Fig. = 1).=20 Besides many novel genes, the list included genes such as VEGF, = IGFBP2,=20 and CHI3L1, which were previously reported as GBM serum = markers.

View this table:
Table 1.=20

GBM-specific potential serum = biomarkers

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Figure 1.=20

One-way hierarchical cluster analysis = of=20 SAM-identified genes using the TMEV software to see the data = substructure.=20 Normalized, log 2=E2=80=93transformed expression ratios of = SAM-identified differentially=20 regulated genes between GBMs (grade IV) and lower-grade astrocytomas = (grades II=20 and III) were subjected to one-way hierarchical clustering using the = TMEV=20 software. A dual-color code was used, with red and green indicating = upregulation=20 and downregulation, respectively, in a particular glioma sample compared = with=20 normal brain tissue. Subclusters A and B represent genes that are = upregulated=20 and downregulated respectively in GBMs compared with lower-grade = astrocytoma=20 (grades II and III).

Validation of GBM-specific secreted biomarkers

Among 34 potential secretory biomarkers that can = discriminate GBM=20 from lower-grade astrocytomas (AA and DA), some of the genes have = already been=20 reported, whereas many were novel and relatively less studied. Some of = the genes=20 that were previously reported, such as CHI3L1, IGFBP2, VEGF, = TNC, and=20 FSTL1, were validated in the present study (data not shown). We = have=20 recently reported FSTL1 as a GBM-specific upregulated marker = (9)= . The=20 novel genes that were validated in this study are CALU, CXCL9, = TIMP1,=20 GPX3, and TIMP3 (Table = 2);=20 other important genes that we have validated are SERPINE1, PLAT, = IL6,=20 and OPN (Table = 2).

View this table:
Table 2.=20

Statistical analysis of regulation of = potential=20 GBM-specific serum biomarkers in different glioma grades

A novel gene upregulated in GBM is calumenin = (CALU), which=20 is a calcium-binding protein located in the endoplasmic reticulum and = involved=20 in protein folding and sorting (14). We=20 found the transcript levels of CALU to be significantly = upregulated to=20 very high levels in GBMs (mean log 2 ratio 1.26 =C2=B1 1.07) compared = with DA, AA,=20 DO, AO, and normal brain samples (mean log 2 ratios =E2=88=920.06 =C2=B1 = 1.01, =E2=88=921.20 =C2=B1 0.71,=20 =E2=88=921.40 =C2=B1 0.78, =E2=88=920.98 =C2=B1 0.88, and 0.00 =C2=B1 = 1.44, respectively; Fig. = 2A; Table = 2).=20 Another interesting gene found upregulated in a majority of GBMs was=20 CXCL9, a member of the chemokine family, which is a group of = small=20 structurally related molecules that regulates cell trafficking of = various types=20 of leukocytes through interactions with a subset of seven-transmembrane, = G=20 protein-coupled receptors. CXCL9 transcripts were found upregulated=20 significantly in GBMs (mean log 2 ratio 3.74 =C2=B1 1.94) compared with = AA, DO, AO,=20 and normal brain samples (mean log 2 ratios 2.10 =C2=B1 2.30, 0.53 = =C2=B1 2.64, 1.28 =C2=B1=20 2.14, and 0.00 =C2=B1 1.91, respectively; Fig. = 2C; Table = 2).

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Figure 2.=20

Scatter plots of the transcript levels = of novel=20 secretory biomarkers, which can potentially discriminate GBM from = astrocytoma=20 grades II/III and oligodendroglioma grades II/III. Log = 2=E2=80=93transformed gene=20 expression ratios obtained from real-time RT-qPCR analysis of RNA = derived from=20 tumor tissue samples (as indicated) are plotted for CALU (A),=20 SERPINE1 (B), CXCL9 (C), TIMP1 (D), = IL6 (E),=20 PLAT (F), GPX3 (G), and TIMP3 (H). Each dot=20 represents a data derived from one sample. In each sample, fold change = in gene=20 expression is calculated over its mean expression in normal brain = samples. An=20 increase or decrease in gene expression at a particular cutoff = (horizontal=20 dotted line) based on the expression pattern of a specific gene across = different=20 grades of tumors was considered significant. Horizontal line in each = group=20 designates the median value.

Two novel genes that were found to be downregulated in GBMs = are=20 GPX3 and TIMP3. GPX3 is a secreted member of=20 glutathione peroxidase family involved in reduction of hydrogen = peroxide,=20 thereby protecting cells from oxidative damage. GPX3 = transcripts were=20 found downregulated significantly in GBMs (mean log 2 ratio 2.20 =C2=B1 = 1.59)=20 compared with AA and normal brain samples (mean log 2 ratios 4.02 =C2=B1 = 1.70 and=20 0.00 =C2=B1 1.63, respectively; Fig. = 2G; Table = 2).=20 However, oligodendroglioma grade II/DO (mean log 2 ratio of 0.99 =C2=B1 = 2.33) and=20 grade III/AO (mean log 2 ratio of 2.27 =C2=B1 1.36) also had an = expression pattern=20 similar to GBM (Fig. = 2G; Table = 2). We=20 also analyzed serum GPX3 protein levels by Western blot (Fig. = 3A).=20 Statistical analysis using Mann-Whitney test revealed that the serum = GPX3=20 protein level, as measured by intensity, in GBM sera (mean intensity = 0.91 =C2=B1=20 0.24) was significantly (P =3D 0.048) less compared with that = of normal=20 sera (mean intensity 1.11 =C2=B1 0.21).

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Figure 3.=20

Analysis of serum TIMP3, GPX3, and = TIMP1. A, total=20 serum protein (5 =CE=BCL) from normal healthy controls and GBM patients = was subjected=20 to Western blot analysis for TIMP3. B, albumin/IgG depleted serum = protein (50=20 =CE=BCg) from normal healthy controls and GBM patients was subjected to = Western blot=20 analysis for GPX3. C, serum levels of TIMP1 (ng/mL) in normal controls=20 (n =3D 34), DAs (n =3D 13), AAs (n =3D 13), = and GBMs=20 (n =3D 52) are plotted. Horizontal line in each group = designates the mean=20 value. Statistical significance was tested by Kruskal-Wallis one-way = ANOVA using=20 SPSS 15.0 for Windows software.

The transcript levels of tissue inhibitor of = metalloproteinase 3=20 (TIMP3) was found to be downregulated significantly in GBMs (mean log 2 = ratio=20 =E2=88=921.53 =C2=B1 1.04) compared with DA, AA, and normal brain = samples (mean log 2 ratios=20 =E2=88=920.31 =C2=B1 0.50, =E2=88=920.60 =C2=B1 0.74, and 0.00 =C2=B1 = 1.57, respectively; Fig. = 2H; Table = 2).=20 However, oligodendroglioma grade 2/DO (mean log 2 ratio of =E2=88=921.22 = =C2=B1 0.72) and=20 grade 3/AO (mean log 2 ratio of =E2=88=921.19 =C2=B1 1.05) also had an = expression pattern=20 similar to GBM (Fig. = 2H; Table = 2). We=20 also analyzed the serum TIMP3 protein levels by Western blot (Fig. = 3B).=20 Statistical analysis using Mann-Whitney test revealed that the serum = TIMP3=20 protein level, as measured by intensity, in GBM sera (mean intensity = 0.67 =C2=B1=20 0.28) was less compared with that of normal sera (mean intensity 1.00 = =C2=B1 0.48),=20 which showed a tendency toward statistical significance (P =3D = 0.068).=20 Thus, these two genes, GPX3 and TIMP3, seem to = discriminate=20 GBM from lower-grade astrocytomas (AA and DA) but not oligodendrogliomas = (AO and=20 DO).

We have also confirmed GBM-specific expression of four more = relatively less studied genes. The first gene is serine proteinase=20 inhibitor, clade E, member 1 (SERPINE1), also known as=20 plasminogen activator inhibitor, type 1 (PAI-1). = SERPINE1 was=20 found to be upregulated significantly in GBMs compared with DA, AA, DO, = and AO=20 (Fig. = 2B; Table = 2). The=20 second gene, TIMP1, is overexpressed significantly in GBMs = compared=20 with DA, AA, DO, AO, and normal samples (Fig. = 2D; Table = 2). We=20 have also validated GBM-specific upregulation of TIMP1 at the level of = serum=20 TIMP1 protein by ELISA (Fig. = 3C).=20 Comparisons of serum TIMP1 levels were made between different grades of=20 astrocytoma and normal sera. Kruskal-Wallis ANOVA followed by post hoc = Tukey's=20 Honest Significant Difference (HSD) test showed that the difference = between GBM=20 and DA and AA to be significant (P < 0.0001 and P = <=20 0.0001, respectively). The mean serum TIMP1 level in the GBM sera was = 159.96 =C2=B1=20 55.40 compared with 61.58 =C2=B1 26.41 in AA and 63.14 =C2=B1 25.45 in = DA. However, the=20 serum TIMP1 levels in normal individuals (mean 173.33 =C2=B1 55.08) was = not different=20 from that of GBM patients. Because the GBM samples used for this = analysis=20 (n =3D 52) underwent uniform treatment protocol and were = prospectively=20 followed up, we analyzed the survival utility of TIMP1. The median = survival=20 among GBM patients in the group with serum TIMP1 levels >195 ng/mL = was less=20 than the group with TIMP1 level lesser than 195 ng/mL (11 months versus = 17=20 months, respectively). However, these data were found to be = statistically not=20 significant (data not shown).

The third gene found to be upregulated in GBMs is = plasminogen=20 activator, tissue type (PLAT; t-PA). PLAT transcripts were = found=20 to be significantly upregulated in GBMs compared with lower-grade = astrocytomas=20 (DA and AA) as well as oligodendrogliomas (DO and AO; Fig. = 2F; Table = 2).=20 Interleukin 6 (IL6) was the fourth gene found to be=20 significantly upregulated in GBMs as against DA, AA, DO, AO, and normal = samples=20 (Fig. = 2E; Table = 2).

We have also measured the transcript levels of these eight = genes in=20 glioma-derived cell lines. In principle, all the eight genes were found = to show=20 similar GBM-specific differential regulation, like GBM tissue samples, = compared=20 with normal brain samples in most glioma cell lines (Supplementary Fig. = S1).=20

OPN is a GBM-specific serum biomarker with prognostic value

Although there were few reports of OPN upregulation in GBM, = we=20 thought that a thorough investigation of OPN regulation in different = grades of=20 glioma is needed. We carried out detailed analysis of OPN at RNA and = protein=20 levels in tumor tissue and serum OPN levels in different grades of = astrocytoma=20 as well as its effect on patient survival. OPN transcript exists in = three=20 isoforms, a, b, and c (corresponding to transcript variants 1, 2, and 3, = respectively), which arises due to differential splicing. All the three = isoforms=20 were found to be upregulated significantly in GBMs compared with DA, AA, = DO, AO,=20 and normal samples, except DA versus GBM in the case of OPN tv1 (Fig. = 4A-C; Table = 2).=20 Interestingly, when glioma-derived cell lines were tested for OPN = transcript=20 levels, we found the OPN transcript variant 2 alone to be upregulated in = most=20 glioma cell lines, whereas the transcript levels of variants 1 and 3 = were=20 actually downregulated in glioma cell lines compared with normal brain = controls=20 (Fig. = 4D-F).=20

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Figure 4.=20

Scatter plot of transcript levels of = OPN in=20 different grades of astrocytoma and glioma cell lines. A to C, log = 2=E2=80=93transformed=20 gene expression ratios obtained from real-time RT-qPCR analysis of RNA = derived=20 from tumor tissue samples (as indicated) are plotted for OPN tv1 = (transcript=20 variant 1; A), tv2 (B), and tv3 (C). Each dot represents a data derived = from one=20 sample. In each sample, fold change in gene expression is calculated = over its=20 mean expression in normal brain samples. An increase or decrease in gene = expression at a particular cutoff (horizontal dotted line) based on the=20 expression pattern of a specific gene across different grades of tumors = was=20 considered significant. Horizontal line in each group designates the = median=20 value. D to F, log 2=E2=80=93transformed gene expression ratios obtained = from real-time=20 RT-qPCR analysis of RNA derived from different glioma cell lines (black = bars,=20 left to right order of cell lines: LN-18, LN-229, U-138, U-251, U-343, = U-373,=20 U-87) are plotted for OPN tv1 (transcript variant 1; D), tv2 (E), and = tv3 (F).=20 The gray bars represent the normal brain control samples used in this=20 experiment. Each bar represents a data derived from one sample. In each = sample,=20 fold change in gene expression is calculated over its mean expression in = normal=20 brain samples.

We also analyzed the OPN protein levels by = immunohistochemistry in=20 astrocytoma tissue sections. We found OPN protein staining to be = cytoplasmic and=20 accentuated at the surface of the tumor cells (Fig. = 5). The=20 macrophages infiltrating the tumor were also stained. In addition to the = tumor=20 cells and macrophages, the interstitial stroma stained prominently in = several=20 examined cases. OPN expression was pronounced around vessels and at the = border=20 of necrosis. Upon semiquantitative analysis of tumor cell positivity of = OPN, we=20 found that a majority of GBM (70%; 33 of 44) had immunopositive cells = compared=20 with AA (23.0%; 6 of 26) and DA (0.0%; 0 of 8). However, staining of the = extracellular matrix was extensive and seen in 95.5% (42 of 44) of GBM = and 50%=20 cases of DA and AA. It was observed that the extent of staining of the=20 extracellular matrix was diffuse and more extensive in GBM tumors = compared with=20 cases of DA or AA (Fig. = 5). In=20 the control samples, there was no staining of neurons, astrocytes,=20 oligodendrocytes, neuropil, and stroma as well as the parenchymal = vessels.

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Figure 5.=20

Immunohistochemical validation of OPN=20 overexpression in astrocytoma. Sections from DA-negative for staining = (A),=20 DA-positive for staining (B), AA=E2=80=94negative for staining (C), = AA=E2=80=94positive for=20 staining (D), and secondary GBMs and primary GBM=E2=80=94positive for = staining (E and F)=20 were stained for OPN.

The levels of OPN protein in serum samples belonging to = different=20 grades of astrocytoma and normal controls were measured by ELISA (Fig. = 6A).=20 Comparisons of serum OPN levels were made between different grades of=20 astrocytoma and normal controls as well as between different grades.=20 Kruskal-Wallis ANOVA followed by post hoc Tukey's HSD test showed the = difference=20 between GBM and normal controls (P =3D 0.001), GBM and DA = (P =3D=20 0.046), and GBM and AA (P =3D 0.008) to be significant. The = mean serum=20 OPN level (ngm/mL) in GBM was 31.54 =C2=B1 28.98 compared with 17.38 = =C2=B1 7.91 in AA,=20 13.79 =C2=B1 4.56 in DA, and 11.70 =C2=B1 7.26 in normal control. We = have also validated=20 serum OPN levels between normal healthy individuals and GBM patients by = Western=20 blot analysis (Fig. = 6B).=20 Statistical analysis using Mann-Whitney test revealed the serum OPN = protein=20 level, as measured by intensity, in GBM sera (mean intensity 1.40 =C2=B1 = 0.43) to be=20 more significant compared with that of normal sera (mean intensity 1.00 = =C2=B1 0.41;=20 P =3D 0.037).

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Figure 6.=20

Scatter plot of serum OPN levels and = its=20 correlation with patient survival. A, serum levels of OPN (ng/mL) in = normal=20 controls (n =3D 22), DAs (n =3D 12), AAs (n = =3D 36), and=20 GBMs (n =3D 78). The horizontal line in each group designates = the mean=20 value. B, total serum protein (2.5 =CE=BCL) from normal healthy controls = and GBM=20 patients was subjected to Western blot analysis for OPN. C, Kaplan-Meier = survival estimates for 30 GBM patients are calculated for their serum = OPN=20 levels. Survival curves for the groups positive and negative for OPN in=20 univariate analysis are shown. Cases that were low for serum OPN levels = (=E2=89=A420=20 ng/mL; black line; n =3D 19) had a better survival than the = cases that=20 were high for serum OPN levels (>20 ng/mL; gray line; n =3D = 11;=20 P =3D 0.0001).

A subset of GBM patients (n =3D 30) who underwent = uniform=20 treatment protocol and were prospectively followed up was used for = survival=20 analysis (Fig. = 6C). The=20 serum OPN level of 20 ng/mL was found to significantly correlate with = the=20 survival. The median survival among GBM patients in the group with serum = OPN=20 levels >20 ng/mL was significantly less than in that group with = =E2=89=A420 ng/mL (9=20 months versus 21 months, respectively; P =E2=89=A4 0.0001; = univariate=20 analysis). On multivariate analysis, taking into consideration other = factors=20 like age, cognitive dysfunction, and p53 status, a serum OPN level = cutoff of 20=20 ng/mL was still independently significant (P =3D <0.0001; = Cox=20 regression analysis, forward Walds). This indicates that the = preoperative serum=20 OPN level is a definite prognostic indicator in GBM.

= Previous=20 SectionNext=20 Section

Discussion

In this study, we have used a unique approach to screen our = cancer=20 microarray database to identify potential secreted biomarkers. In the = first=20 step, we pulled out expression data of all the genes whose protein = products=20 localized either in the plasma membrane or were secreted. Using SAM, we=20 identified GBM-specific serum markers and subsequently validated many of = them.=20 Some of the genes identified in this screen, such as CHI3L, = IGFBP2, and=20 VEGFA, have been previously reported as GBM-specific serum = markers with=20 prognostic value (15-18). In=20 our study, we could also show the GBM-specific expression of the = transcripts of=20 these genes by RT-qPCR (data not shown). These previous findings from = others and=20 the real-time RT-qPCR validation from our study provide support to our=20 methodology of detecting serum biomarkers.

We have also identified few novel genes that are = specifically=20 regulated in GBM. CALU is a novel gene found upregulated = specifically=20 in GBM but not in low-grade astrocytomas and oligodendrogliomas. CALU = belongs to=20 a family of multiple EF-hand proteins that include reticulocalbin, = ERC-55, and=20 Cab45, and human CALU has been shown to be secreted (19).=20 However, the significance of upregulation of CALU in GBMs is not known. = CXCL9, a=20 member of chemokine family, was found in this study as an upregulated=20 GBM-secreted biomarker. Although CXCL9 and other members have been shown = to be=20 upregulated in other cancers, we report for the first time that CXCL9 is = a=20 GBM-specific secretory marker (20, 21). It is=20 interesting to note that although our microarray data had other CXCL = genes,=20 CXCL10, CXCL12, and CXCL13, only CXCL9 was = identified=20 by SAM analysis, suggesting the specific upregulation of CXCL9 from the=20 chemokine family in GBMs.

We also identified two genes, GPX3 and = TIMP3,=20 specifically downregulated in GBMs compared with lower-grade = astrocytomas (DA=20 and AA) but not in oligodendrogliomas (DO and AO). We show our initial=20 microarray data=E2=80=93based finding that these two genes are = downregulated in GBM by=20 ELISA and Western blot. GPX3 is also called as extracellular or plasma=20 glutathione peroxidase and is the only member of glutathione peroxidase = family=20 that is secreted. Although our microarray data had other members of the = GPX=20 family, GPX1, GPX4, and GPX6, only GPX3 was=20 identified by SAM, which showed specific upregulation in GBMs. These two = markers, GPX3 and TIMP3, are of value in = differentiating=20 oligodendroglioma grade 2 and 3 from astrocytoma grade 2 and 3, as these = markers=20 were overexpressed in both DO and AO (Fig. 2G = and H;=20 Table = 2).

TIMP1 and TIMP3, members of the tissue inhibitor of = metalloproteases,=20 were found differentially regulated in GBMs. Matrix metalloproteinases, = many of=20 which are overexpressed in malignant tumors, including GBM, are = inhibited by=20 TIMPs. It is suggested that a balance between the matrix = metalloproteinases and=20 TIMPs may contribute to malignant behavior of many cancers. Although = TIMPs are=20 known for their inhibition of tumor malignancy, a differential role for = some of=20 TIMPs has been reported. For example, TIMP1 has been shown to stimulate=20 proliferation of many cell types (22-25). In=20 good correlation, our data clearly provide evidence for the GBM-specific = upregulation of TIMP1. We have found that TIMP1 transcripts were = upregulated=20 severalfold in GBMs compared with other grades of astrocytoma and=20 oligodendroglioma (Fig. = 2D). In=20 addition, serum TIMP1 levels are elevated in GBM patients compared with=20 lower-grade astrocytoma patients (Fig. = 3C).=20 Unlike TIMP1, TIMP3 has been shown to promote apoptosis possibly through = stabilization of tumor necrosis factor =CE=B1 receptors (26-28).=20 Accordingly, we found TIMP3 transcripts downregulated significantly in = GBMs=20 compared with lower-grade astrocytomas (Fig. = 2H).

Another interesting gene we have validated was = OPN, which is=20 a member of the SIBLING (small integrin-binding ligand N-linked = glycoprotein)=20 family and has been found to be overexpressed in many cancers and = associated=20 with tumor progression and metastasis (29-32). High=20 levels of OPN expression correlate with tumor invasion, progression, or=20 metastasis in multiple cancers. Studies show that OPN mediates the = molecular=20 mechanisms that determine metastatic spread, such as prevention of = apoptosis,=20 extracellular matrix proteolysis and remodeling, cell migration, evasion = of=20 host-immune cells, and neovascularization. Transcriptional regulation of = OPN is=20 complex and involves multiple pathways, including AP-1, Myc, v-Src, = Runx/CBF,=20 TGF-=CE=B2/BMPs/Smad/Hox, and Wnt/=CE=B2-catenin/APC/GSK-3=CE=B2/Tcf-4 = (29). OPN=20 expression has been found to correlate with tumor progression in = astrocytoma=20 with high levels in GBM (33-35). OPN=20 levels detected by immunohistochemistry identified that high cytoplasmic = OPN=20 expression correlates with poor patient survival (36).=20 Although serum OPN levels have been shown to correlate with patient = survival in=20 other cancers (37), there=20 has been no such report with respect to glioma. Our results show that = OPN is=20 upregulated both at the RNA and protein levels only in GBM. Further, = serum OPN=20 levels were high in GBM and are poor prognostic indicators. Our findings = are=20 also supported by recently published TCGA data, in which we found that = seven of=20 nine genes (CALU, CXCL9, IL6, PLAT, TIMP1, OPN, and = SERPINE1)=20 are similarly regulated (38). Thus,=20 we provide evidence for analyzing large microarray data through = bioinformatics=20 methods to find potential serum/secreted biomarkers.

Previous=20 Section= Next=20 Section

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were = disclosed.

Previous=20 Section<= SPAN>Next=20 Section

Acknowledgments

We thank Shailaja Chandrashekararao, Cini Samuel, K. = Premkumar, and=20 K. Chandrasekar. Infrastructural support by funding from Indian Council = of=20 Medical Research (Center for Advanced Studies in Molecular Medicine), = Department=20 of Biotechnology, Department of Science and Technology (Fund for = Improvement of=20 S$T Infrastructure), and University Grants Commission to the Department = of MCB,=20 Indian Institute of Science, is acknowledged.

Grant Support: Grant from CSIR, Government = of India,=20 under the NMITLI program. K. Somasundaram is a Wellcome Trust = International=20 Senior Research Fellow.

The costs of publication of this article were defrayed in = part by the=20 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.

= Previous=20 Section<= SPAN>Next=20 Section

Footnotes

  • Note: Supplementary data for this article = are=20 available at Cancer Epidemiology, Biomarkers & Prevention Online=20 (http://cebp.aacrjournals.org/).

  • Received October 15, 2009.
  • Revision received March 18, 2010.
  • Accepted March 31, 2010. =

<= SPAN>Previous=20 Section

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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 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tempClassArr[i].replace(/^x-/,''));=0A= }=0A= }=0A= }=0A= linkContents =3D tempSpan.html().replace(/{at}/,'@');=0A= newHtml =3D '' + linkContents + '';=0A= }=0A= else {=0A= linkContents =3D addr;=0A= }=0A= }=0A= if (newHtml =3D=3D '') {=0A= newHtml =3D '' + linkContents + '';=0A= }=0A= $(this).replaceWith(newHtml);=0A= }=0A= }=0A= );=0A= =0A= /* make links with rel=3D"external-nw" target a new window */=0A= $("a[rel*=3Dexternal-nw]").each(=0A= function(i) {=0A= var id =3D $(this).attr("id");=0A= var targetName =3D "_blank";=0A= var config =3D null;=0A= if ((id !=3D undefined) && id && !(id =3D=3D '') &&=0A= !(gSiteOptions.openWindowDetails =3D=3D undefined)) {=0A= var newLinkOptions =3D gSiteOptions.openWindowDetails[id];=0A= if ((newLinkOptions !=3D undefined) && newLinkOptions) {=0A= var overrideTarget =3D newLinkOptions.target;=0A= var overrideConfig =3D newLinkOptions.config;=0A= if ((overrideTarget !=3D undefined) && overrideTarget && = (overrideTarget.length > 0)) {=0A= targetName =3D overrideTarget;=0A= }=0A= if ((overrideConfig !=3D undefined) && overrideConfig && = (overrideConfig.length > 0)) {=0A= config =3D overrideConfig;=0A= }=0A= }=0A= }=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", targetName).attr("title", newTitle);=0A= if (config !=3D null) {=0A= $(this).click(function() {=0A= window.open(this.href, targetName, config);=0A= return false;=0A= });=0A= }=0A= }=0A= );=0A= =0A= /* attach onFocus handlers to quick-search fields, to clear the=0A= field when it receives focus IF it contains the default=0A= value (such as "Search the site") */=0A= updateFormInput("#header-qs-search-label", "#header-qs-input", '', '');=0A= =0A= /* handle collapsing content-box areas, with user prefs */=0A= setupCollapsibles();=0A= =0A= /* handle "most" boxes */=0A= handleMostBoxes();=0A= });=0A= =0A= =0A= function setupCollapsibles() {=0A= // sites can override this if they want to target different elements=0A= prepCollapsibles("div.content-box div.collapsible");=0A= }=0A= =0A= /* handle collapsing content-box areas, with optional user prefs */=0A= function prepCollapsibles(parentJQueryEl, setPrefs) {=0A= if (typeof(setPrefs) =3D=3D "undefined") {=0A= setPrefs =3D true;=0A= }=0A= var expandStr, contractStr;=0A= if (gSiteOptions =3D=3D undefined) {=0A= expandStr =3D '+';=0A= contractStr =3D '-';=0A= }=0A= else {=0A= if (gSiteOptions.cbExpandString !=3D undefined) {=0A= expandStr =3D gSiteOptions.cbExpandString;=0A= }=0A= else if (gSiteOptions.expandString !=3D undefined) {=0A= expandStr =3D gSiteOptions.expandString;=0A= }=0A= else { expandStr =3D '+'; }=0A= if (gSiteOptions.cbContractString !=3D undefined) {=0A= contractStr =3D gSiteOptions.cbContractString;=0A= }=0A= else if (gSiteOptions.contractString !=3D undefined) {=0A= contractStr =3D gSiteOptions.contractString;=0A= }=0A= else { contractStr =3D '-'; }=0A= }=0A= $(parentJQueryEl).each(=0A= function(i) {=0A= var $this =3D $(this);=0A= var id =3D $this.attr("id");=0A= if (id !=3D null) {=0A= var $labelElement, $label;=0A= for (var i =3D 0; i < gSiteOptions.collapsibleLabels.length; i++) {=0A= $labelElement =3D gSiteOptions.collapsibleLabels[i];=0A= $label =3D $this.find($labelElement);=0A= if ($label.length) {=0A= // get first one=0A= if ($label.length > 1) {=0A= $label =3D $label.eq(0);=0A= }=0A= break;=0A= }=0A= }=0A= =0A= // if there's already a toggle leave this as is=0A= var alreadyHasToggle =3D ($label.length && = $label.find("a.collapse-toggle").length);=0A= if ($label.length) {=0A= if (!(alreadyHasToggle)) {=0A= var labelText =3D $label.html();=0A= $label.empty().append('' + contractStr + = ' ' + labelText + '');=0A= }=0A= }=0A= =0A= var isOpen =3D true;=0A= if (setPrefs) {=0A= var statePref =3D getPrefValue(id + "-state");=0A= if (statePref && ((statePref =3D=3D 'open') || (statePref =3D=3D = 'closed'))) {=0A= if (statePref =3D=3D 'closed') {=0A= isOpen =3D false;=0A= }=0A= }=0A= else {=0A= // no pref, or unknown value=0A= if ($this.hasClass('default-closed')) {=0A= isOpen =3D false;=0A= }=0A= }=0A= }=0A= var childListType;=0A= if ($this.children("ol").length) {=0A= childListType =3D "ol";=0A= }=0A= else {=0A= childListType =3D "ul";=0A= }=0A= if (!isOpen && !(alreadyHasToggle)) {=0A= toggleCollapse($this, childListType, ($labelElement + " a = span.view-more"), expandStr, contractStr);=0A= }=0A= $this.find("a.collapse-toggle").click(=0A= function(e) {=0A= // need to find first parent that is in either state...=0A= // $(this).parents(".collapsible, .collapsed").each(=0A= $(this).parents().each(=0A= function(i) {=0A= if ($(this).hasClass('collapsible') || = $(this).hasClass('collapsed')) {=0A= var isCollapsed =3D $this.hasClass('collapsed');=0A= if (setPrefs) {=0A= var prefName =3D id + "-state";=0A= if (isCollapsed) {=0A= setPref(prefName, "open");=0A= }=0A= else {=0A= setPref(prefName, "closed");=0A= }=0A= }=0A= toggleCollapse($(this), childListType, ($labelElement + " a = span.view-more"), expandStr, contractStr);=0A= fixColHeights(-1);=0A= return false;=0A= }=0A= }=0A= );=0A= e.preventDefault();=0A= }=0A= );=0A= }=0A= }=0A= );=0A= }=0A= =0A= function handleMostBoxes() {=0A= var hadMostBoxes =3D false;=0A= $("div.most-links-box").each(=0A= function(i) {=0A= var $this =3D $(this);=0A= modClass($this, '', 'js-marker');=0A= hadMostBoxes =3D true;=0A= // 1) get the
    child=0A= var $mostlist =3D $this.children("ul");=0A= // 2) build a new
      for the header, from

      child:=0A= var hasSelected =3D false;=0A= var mostHdrList =3D null;=0A= if ($mostlist.length) {=0A= var hasItems =3D false;=0A= var hdrList =3D '
        ';=0A= var $mostli =3D $mostlist.children("li");=0A= if ($mostli.length) {=0A= hasItems =3D true;=0A= $mostli.each(=0A= function(i) {=0A= var $hdr =3D $(this).children("h4").html();=0A= if ($(this).hasClass("most-cur-sel")) {=0A= hasSelected =3D true;=0A= hdrList +=3D '
      • ' + $hdr + '
        • ';=0A= }=0A= else {=0A= hdrList +=3D '
      • ' + $hdr + '
        • ';=0A= }=0A= }=0A= );=0A= }=0A= hdrList +=3D '
      ';=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= addDockBlockCallback(dockId);=0A= }=0A= else if (gNavDocked && (navBottom > 0)) {=0A= /* undock the nav */=0A= gNavDocked =3D false;=0A= removeDockBlock(col, dockId);=0A= removeDockBlockCallback(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= // custom implementations can override this function to do something = when a dockBlock is added=0A= // they should return false if they want to stop any additional default = handling=0A= function customAddDockBlockCallback(dockId) {=0A= return true;=0A= }=0A= // this function should not be overridden=0A= function addDockBlockCallback(dockId) {=0A= if (customAddDockBlockCallback(dockId) !=3D false) {=0A= // no default handling, currently=0A= }=0A= }=0A= // custom implementations can override this function to do something = when a dockBlock is removed=0A= // they should return false if they want to stop any additional default = handling=0A= function customRemoveDockBlockCallback(dockId) {=0A= return true;=0A= }=0A= // this function should not be overridden=0A= function removeDockBlockCallback(dockId) {=0A= if (customRemoveDockBlockCallback(dockId) !=3D false) {=0A= // no default handling, currently=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; i < prefArray.length; i++) {=0A= if (prefArray[i].indexOf(match) =3D=3D 0) {=0A= return prefArray[i].substring(match.length);=0A= }=0A= else if (prefArray[i] =3D=3D name) {=0A= // name only, no value, return boolean true=0A= return true;=0A= }=0A= }=0A= }=0A= return null;=0A= }=0A= function convertPrefString(str) {=0A= if (str) {=0A= return str.replace(/[,:=3D]/g,"");=0A= }=0A= return str;=0A= }=0A= function removePref(name) {=0A= var prefArray =3D getPrefArray();=0A= name =3D convertPrefString(name);=0A= if (prefDefined(name)) {=0A= var newArray =3D new Array(prefArray.length - 1);=0A= var match =3D name + ":";=0A= var newArrIndex =3D 0;=0A= for (var i =3D 0; i < prefArray.length; i++) {=0A= if (!((prefArray[i].indexOf(match) =3D=3D 0) || (prefArray[i] =3D=3D = name))) {=0A= newArray[newArrIndex++] =3D prefArray[i];=0A= }=0A= }=0A= setPrefCookie(newArray);=0A= if (prefArray.length =3D=3D 1) {=0A= return null;=0A= }=0A= else {=0A= return newArray;=0A= }=0A= }=0A= else {=0A= return prefArray;=0A= }=0A= }=0A= =0A= function setPref(name, value) {=0A= if (typeof(value) =3D=3D "undefined") {=0A= value =3D null;=0A= }=0A= if (name) {=0A= name =3D convertPrefString(name);=0A= var prefArray =3D removePref(name);=0A= if (prefArray =3D=3D null) {=0A= prefArray =3D new Array;=0A= }=0A= var newArray =3D new Array(prefArray.length + 1);=0A= var newPref =3D name + (((value !=3D null) && (value !=3D '')) ? (':' = + value) : '');=0A= newArray[0] =3D newPref;=0A= for (var i =3D 0; i < prefArray.length; i++) {=0A= newArray[i+1] =3D prefArray[i];=0A= }=0A= setPrefCookie(newArray);=0A= return newArray;=0A= }=0A= else {=0A= return getPrefArray();=0A= }=0A= }=0A= function setPrefCookie(prefs) {=0A= if (prefs && (prefs.length > 0)) {=0A= var prefString;=0A= if (typeof(prefs) =3D=3D 'string') {=0A= prefString =3D prefs;=0A= }=0A= else {=0A= if (prefs.length =3D=3D 1) {=0A= prefString =3D prefs[0];=0A= }=0A= else {=0A= prefString =3D prefs.join(',');=0A= }=0A= }=0A= setCookie(UIPrefsCk.name, prefString, UIPrefsCk.expDays, = UIPrefsCk.path, false, false);=0A= }=0A= else {=0A= deleteCookie(UIPrefsCk.name, UIPrefsCk.path, false);=0A= }=0A= }=0A= // individual pref items in the cookie are separated by ',' and=0A= // nv pref pairs are joined by ':', i.e. n1:v1,n2:v2,n3:v3,etc.=0A= // values are optional. (i.e. "n1,n2:v2,n3" is ok)=0A= function getPrefArray() {=0A= var cookieVal =3D getCookie(UIPrefsCk.name);=0A= if (cookieVal !=3D null) {=0A= return cookieVal.split(',');=0A= }=0A= return null;=0A= }=0A= =0A= // http://www.dustindiaz.com/top-ten-javascript/=0A= function getCookie( name ) {=0A= var start =3D document.cookie.indexOf( name + "=3D" );=0A= var len =3D start + name.length + 1;=0A= if (((!start ) && (name !=3D document.cookie.substring( 0, name.length = ))) || (start =3D=3D -1)) {=0A= return null;=0A= }=0A= var end =3D document.cookie.indexOf( ';', len );=0A= if ( end =3D=3D -1 ) end =3D document.cookie.length;=0A= return unescape( document.cookie.substring( len, end ) );=0A= }=0A= function setCookie( name, value, expires, path, domain, secure ) {=0A= var today =3D new Date();=0A= today.setTime( today.getTime() );=0A= if ( expires ) {=0A= expires =3D expires * 1000 * 60 * 60 * 24;=0A= }=0A= var expires_date =3D new Date( today.getTime() + (expires) );=0A= document.cookie =3D name+'=3D'+escape( value ) +=0A= ( ( expires ) ? ';expires=3D'+expires_date.toGMTString() : '' ) +=0A= ( ( path ) ? ';path=3D' + path : '' ) +=0A= ( ( domain ) ? ';domain=3D' + domain : '' ) +=0A= ( ( secure ) ? ';secure' : '' );=0A= }=0A= =0A= function deleteCookie( name, path, domain ) {=0A= if ( getCookie( name ) ) document.cookie =3D name + '=3D' +=0A= ( ( path ) ? ';path=3D' + path : '') +=0A= ( ( domain ) ? 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      This Article

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        ' + expandStr + ' Author Affiliations

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'&' : = '?') + '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= 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; 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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; 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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"); 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        Not = available in this view
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        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
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        ';=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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