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Neoplasia. 2006 December; = 8(12):=20 1072=E2=80=931082.
PMCID:=20 = PMC1790729
Copyright=20 =C2=A9 2006 Neoplasia Press, Inc. All rights reserved
Notch Signaling Enhances Nestin Expression = in=20 Gliomas1*
Alan H Shih* and = Eric C=20 Holland*=E2=80=A0
*Department of Cancer Biology and = Genetics,=20 Memorial Sloan-Kettering Cancer Center, New York, NY 10021, = USA
=E2=80=A0Department of Surgery = (Neurosurgery),=20 Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY = 10021,=20 USA
Address all = correspondence to:=20 Eric C. Holland, 1275 York Avenue, New York, NY 10021. E-mail: = hollande/at/mskcc.org
Received July 28, 2006; Revised September = 28, 2006;=20 Accepted October 2, 2006.
3D"Small = This=20 article has been cit= ed=20 by other articles in PMC.
Abstract
Recent findings = suggest that=20 Notch signaling is active in brain tumors and stem cells and that = stem=20 cells or cells with progenitor characteristics contribute to brain = tumor=20 formation. These stem cells are marked by expression of several = markers=20 including nestin, an intermediate filament protein. We have = studied how=20 the Notch signaling pathway affects nestin expression in brain = tumors. We=20 find that Notch receptors and ligands are expressed in = vitro and=20 in human samples of glioblastomas, the highest grade of malignant = gliomas.=20 In culture, Notch activity activates the nestin promoter. = Activation of=20 the Notch pathway also occurs in a glioblastoma multiforme mouse = model=20 induced by Kras, with translational regulation playing a role in = Notch=20 expression. Combined activation of Notch and Kras in wild-type=20 nestin-expressing cells leads to their expansion within the = subventricular=20 zone and retention of proliferation and nestin expression. = However,=20 activation of Notch alone is unable to induce this cellular = expansion.=20 These data suggest that Notch may have a contributing role in the=20 stem-like character of glioma cells.
Keywords: Glioma, nestin, mouse model, Notch, stem=20 cell
Introduction
Among tumors of the = central nervous=20 system (CNS), glioblastoma multiformes (GBMs) are the most = aggressive=20 tumors with the poorest clinical prognosis. These tumors consist = of cells=20 that are astrocyte-like but have complex genetic makeup and = expression=20 patterns. One phenomenon that can contribute to this complexity is = the=20 presence of stem-like cells within the tumor and the activation of = pathways that control cellular differentiation. Several groups = have been=20 able to identify unique populations within GBMs with stem cell = properties=20 [1=E2=80=933]. However, which pathways = contribute to the=20 formation of stem-like character and to the dysregulation of=20 differentiation remain to be fully described. One marker that has = been=20 used to identify neural stem cells is the intermediate filament = protein=20 nestin [4]. Nestin-expressing cells have the ability to = differentiate=20 into multiple lineages, including neurons and glial cells within = the CNS=E2=80=94a=20 characteristic found in glioma stem cells [5].
In trying to understand signaling = events that=20 regulate GBMs, work in our laboratory has previously identified = the Ras=20 and Akt pathways as being elevated in human GBMs, with a unique=20 synergistic effect on the translation of a subset of mRNA [6]. One such target of increased translation is = Notch1 mRNA.=20 Notch (Notch1=E2=80=934 in mammals) is a family of transmembrane = receptors that=20 regulate cell-cell signaling (reviewed in Artavanis-Tsakonas et = al. [7]). Notch ligands (Delta-like1, Delta-like3, and=20 Delta-like4, and Jagged1=E2=80=932 in mammals) are also = transmembrane proteins=20 and, when bound to Notch, act to expose the receptor to = proteolytic=20 activation. Presenilins cleave Notch to generate a Notch1 = intracellular=20 domain (NICD), which then translocates to the nucleus to act as a=20 transcriptional activator [8].
Activated Notch signaling appears = particularly=20 capable of affecting both tumorigenesis and stem cell development. = Notch=20 signaling was first shown to be inhibitory toward neurogenesis and = essential in maintaining a pool of undifferentiated stem cells [9,10]. Deletion of Notch1 results in = reduction=20 in neural stem cells; in presenilin-/- mutants = defective in Notch signaling, neural stem cells have reduced = proliferative=20 capacities [11]. Later studies have expanded the function of = Notch to=20 involve important cell fate decisions throughout glial and = neuronal=20 development. In the oligodendrocyte lineage, Notch activation has = been=20 shown to suppress terminal oligodendrocyte differentiation but = also to=20 support the specification of oligodendrocyte precursors from the = initial=20 stem cell pool [12,13]. Notch signaling can promote the = specification of=20 Muller glia in the rat retina [14,15], radial glia in the telencephalon from = cortical=20 stem cells [16], and astrocytes in the adult brain from = hippocampal=20 multipotent progenitors [17].
Notch signaling has been = implicated in various=20 steps throughout tumorigenesis. Studies have identified the Ras = pathway,=20 in particular, as being able to collaborate with the Notch pathway = to=20 maintain and establish neoplastic phenotype [18,19]. In CNS tumors, Notch signaling = components were=20 found to be deregulated in meningiomas [20], and its activity is observed to be critical = in=20 medulloblastomas [21,22]. In gliomas, Notch1 and its ligands = Delta-like1,=20 Delta-like3, and Jagged1 have also been shown to be involved in = glioma=20 cell survival, differentiation, and proliferation [23,24].
In this study, we = investigated the=20 role of Notch activation in glioblastoma biology. We confirmed = that Notch=20 receptors and ligands are expressed in human GBMs. This was also = true in a=20 Kras-induced mouse GBM model. In these tumors, Notch may = participate in=20 the transcription of specific gene targets, particularly that of = nestin, a=20 neural progenitor marker. The activation of nestin promoter by = Notch was=20 also seen in cultures where Notch can directly act on the=20 nestin-regulatory region to activate its transcription. In = vivo,=20 we found that combined activation of Notch and Kras signaling was=20 sufficient to generate lesions along the subventricular zone = (SVZ). These=20 lesions expressed nestin and a marker of proliferation, suggesting = that=20 they may be early precursor lesions to = tumorigenesis.
Materials and Methods
Tumor Samples
All human tissues = were=20 collected by the Memorial Sloan-Kettering Institute tissue bank,=20 snap-frozen, and stored at -80=C2=B0C. Samples in liquid nitrogen = were ground=20 in mortar and pestle. Protein was extracted from the powder = through lysis=20 with T-per tissue extraction solution (Pierce Biochemical, = Rockford, IL)=20 supplemented with miniTab protease inhibitors (Boehringer = Mannheim,=20 Ingelheim, Germany), 30 mM sodium fluoride, and 1 mM sodium=20 vanadate.
Microarray
Mouse tumors and=20 arf-/- cortex were dissected from brains and frozen in = liquid=20 nitrogen. Tissue in liquid nitrogen was ground to powder form by = mortar=20 and pestle. Probe preparation and hybridization to chips were = performed as=20 described before [6]. Briefly, RNA was prepared using Trizol = (Invitrogen,=20 Carlsbad, CA) and Qiagen RNA Easy kit (Qiagen, Valencia, CA). cRNA = was=20 generated by in vitro transcription using standard = procedures.=20 Probes were hybridized to Affymetrics MOE430 chip (Affymetrics, = Santa=20 Clara, CA). Chips were scanned by a Hewlett Packard GeneArray = Scanner=20 (Hewlett Packard, Palo Alto, CA), and data were collected by = MicroArray=20 Suite Software (Affymetrics). 5=E2=80=B2 Upstream promoter = sequences were derived=20 from ensembl.org.
Western Blot Analysis
Proteins were = separated on=20 sodium dodecyl sulfate-polyacrylamide gel electrophoresis and = transferred=20 to nitrocellulose membranes. The blocking reagent was 5% nonfat = dry milk=20 in phosphate-buffered saline (PBS)-0.1% Tween 20. Primary and = secondary=20 antibodies were prepared in the same blocking reagent. Horseradish = peroxidase (HRP)-conjugated secondary antibodies were used at = 1:1000=20 dilution and visualized using ECL chemiluminescence (Amersham, = Piscataway,=20 NJ), hNotch1 1:50 [bTAN20; Developmental Studies Hybridoma Bank = (DSHB)=20 (Iowa City, IA)], hNotch2 1:50 (C651.6DbHN; DSHB), mNotch1 1:500=20 (Neomarkers, Fremont, CA), Delta-like1 1:500 (Santa Cruz = Biotechnology,=20 Santa Cruz, CA), Jagged1 1:500 (Santa Cruz Biotechnology), actin = 1:1000=20 (Santa Cruz Biotechnology), anti-mouse HRP (Boehringer Mannheim), = and=20 anti-rabbit HRP (Amersham).
DNA Plasmids
The construction of = the=20 retroviral vectors RCAS-Kras and RCAS-Akt has been previously = described=20 [25,26]. The RCAS(B) mouse Notch1 ICD plasmid = was=20 provided by Dr. David Anderson and subcloned into RCAS(A). The = RCAS(B)=20 control virus consists of noncoding (empty) or nonproductive virus = plasmids. Rat Delta1 and rat Jagged1 cDNA were provided by Dr. = Gerry=20 Weinmaster. The nestin-tk reporter plasmid was provided by Dr. = Urban=20 Lendahl [27,28].
Infection of tv-a Transgenic = Mice and=20 Tissue Processing
Generation of the = transgenic=20 mouse line that expresses the RCAS receptor from the nestin = promoter=20 nestin tv-a (Ntv-a) and Ntv-a arf-/- mice has been = previously=20 described [25,29]. Neonatal mice were injected in the = cerebral=20 hemisphere with DF-1 chicken fibroblasts, producing appropriate = RCAS=20 viruses on postnatal day 1. Mice were monitored and then = sacrificed when=20 symptomatic or at the end of the study. Brains were fixed in = formalin,=20 processed, embedded in paraffin, and sectioned as previously = described [30]. Chi-square analysis was used to determine = statistical=20 significance.
In Situ = Hybridization
Probe fragments were = cloned into=20 pDrive Cloning Vector (Qiagen) using polymerase chain reaction = (PCR) or=20 restriction digest. Probe lengths correspond to the following = stretches of=20 cDNA, as demarcated by the following 5=E2=80=B2 and 3=E2=80=B2 = sequences:
Notch probe: 5=E2=80=B2seq CAGCATGGCCAGCTCTGGTT, = 3=E2=80=B2seq=20 AGCAGCATCCACATTGTTCA
Hes1 probe: 5=E2=80=B2seq ATGCCAGCTGATATAATGGA, = 3=E2=80=B2seq=20 TCAGTTCCGCCACGGCCTCC
rDelta1 probe: 5=E2=80=B2seq ATCACACCTGGAGCCGAGAG, = 3=E2=80=B2seq=20 GGCCGCTACTGTGAAGGTCC
rJagged1 probe: 5=E2=80=B2seq GGCCGGGGCGCCCCTTGAGC, = 3=E2=80=B2seq=20 GGCTGGGGTTTATCATGCCT.
Radioactive sense and = antisense=20 probes incorporating [32P]UTP were prepared using T7 or = SP6=20 polymerase. Tissue sections were deparaffinized and treated with=20 proteinase K. Slides were prehybridized with salmon sperm DNA and=20 hybridized overnight with probe in hybridization buffer (50% = formamide,=20 0.3 M NaCl, 20 mM Tris pH 8.0, 5 mM EDTA, 10 mM Na-phosphate, 10% = dextran=20 sulfate, 1x Denhardt's solution, and 500 =C2=B5g/ml yeast tRNA) at = 65=C2=B0C. Slides=20 were washed, dipped in emulsion, exposed for 2 to 4 weeks, and = developed.=20 Three to five tumors of each type were analyzed with each probe=20 set.
Image Analysis
For signal density = analysis,=20 paired dark-field images of normal cortex and tumor regions were = taken=20 from the same slide using Zeiss Axioplan microscopes (Zeiss, = Oberkochen,=20 Germany). All images were taken at fixed exposure and resolution. = Ten such=20 paired images were generated for each tumor type. Images were then = processed on Metamorph software (Metamorph, Sunnyvale, CA) to = determine=20 the area of positive signal pixels. Areas of paired images were = compared=20 (tumor to normal tissue), and statistical significance was = determined=20 using t-test.
Immunohistochemistry = (IHC)
Sections were = processed for IHC=20 as described previously, with antigen retrieval in citrate buffer = with=20 steam [31]. Antibody detection was visualized with = peroxidase and=20 DAB processing (Vector, Burlingame, CA). The antibodies used are = as=20 follows: 9E10 1:200 (human c-myc; Covance, Princeton, NJ), nestin = 1:1000=20 (PharMingen, San Diego, CA), and proliferating cell nuclear = antigen (PCNA)=20 1:1000 (Chemicon, Temecula, CA).
Luciferase Assay
Cells were = transfected with=20 plasmid containing pHES1-luciferase, pCMV-RL (Promega, Madison, = WI), and=20 RCAS expression vector (control, Notch, or Delta) using Fugene = (Roche,=20 Nutley, NJ) according to the manufacturer's instructions. Lysates = were=20 prepared, and luminescence was determined according to the Promega = Dual-Luciferase Reporter System protocol (Promega). Readings were=20 performed with a Turner Biosystems platereader. Firefly = luminescence was=20 normalized to renilla luminescence. Hes1 reporter was subjected to = PCR=20 with genomic DNA using the primers 5=E2=80=B2seq = ACGGGGTACCCTCAGGCGCGCGCCATTGGCC=20 and 3=E2=80=B2seq CCGAAGATCTGCTTACGTCCTTTTACTTGAC. The fragment = contains suspected=20 CBF-1-binding sites. The fragment was cloned into the PGL3-Basic = Vector=20 (Promega) using KpnI and BglII.
=CE=B2-Galactosidase = Assay
Cells were = transfected with=20 =CE=B2-galactosidase reporter constructs using Fugene according to = manufacturer's instructions. Two days after transfection, cells = were fixed=20 in neutral-buffered formalin and washed in PBS. Cells were then = incubated=20 at 37=C2=B0C with a solution containing 1 mg/ml X-gal, 20 mM=20 K3Fe(CN)6, and 2 mM MgCl2 for 6 = hours=20 until a blue precipitate was visible.
Results
Presence of Notch Receptors and = Ligands=20 in Primary Human Glioblastomas
To determine whether = Notch signaling=20 could contribute to gliomas and GBMs in particular, we compared = astrocytic=20 mixed astrocytic gliomas (World Health Organization grades II and = III),=20 GBM expression profiles, and protein levels. Previous work in our=20 laboratory has described a microarray experiment that identified = genes=20 overexpressed in human gliomas [32]. This experiment compared 9 GBMs and 10 = non-GBM gliomas=20 to pooled normal brain. Using this database, we found that five of = nine=20 GBMs had a =E2=89=A5 2-fold expression in Jagged1 mRNA compared to = normal brain,=20 whereas none of the non-GBM gliomas had any appreciable difference = in=20 Jagged1 expression (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 1A3D"Figure).=20 Unfortunately, the probes for Notch1 and Delta-like1 were not = represented=20 on the array used at the time. This suggested that Notch ligand = expression=20 may contribute to glioblastoma formation.
To determine the protein = levels of=20 Notch receptors and ligands in human tumors, a selection of = primary human=20 tumors (GBMs and non-GBM gliomas) was subjected to Western blot = analysis=20 (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 1B3D"Figure).=20 We found Notch1 to be expressed in all tumor types examined, = whereas=20 Notch2 was expressed in a subset of tumors. Notch ligands = Delta-like1 and=20 Jagged1 were more selectively expressed in GBMs. The coexpression = of Notch=20 and its ligands demonstrates that autocrine or juxtacrine modes of = activation are possible, particularly in GBMs and but perhaps also = in a=20 smaller subset of glial tumors.
Expression of Notch Receptors = and Ligands=20 in Mouse Models of Tumors
Given the finding in = human tumors,=20 we investigated whether this pattern of expression was also found = in mouse=20 models of glial tumors, particularly comparing mouse models of = GBMs and=20 oligodendrogliomas, a clinically less aggressive form of glioma. = In=20 addition, because we know that translation may play an important = role in=20 GBMs, we assessed differences in expression in both mRNA and = protein=20 levels. Previously, we have developed various mouse models of CNS = tumors=20 generated through the RCAS/tv-a system. In this system, tv-a = receptor is=20 expressed from a selective promoter by a transgene, whereas an = RCAS virus=20 delivers oncogenes only into these targeted cells [33]. Two tv-a transgenic lines have been used in = glioma=20 modeling studies. One expresses tv-a from the nestin promoter = (Ntv-a),=20 whereas the other expresses tv-a from GFAP promoter (Gtv-a). The = cell=20 specificity of gene transfer mediated by RCAS infection in these = mice is=20 determined by promoter-driven tv-a expression. After infection, = oncogenes=20 are driven from the RCAS viral promoter and are unaffected by the = activity=20 of the promoter driving the tv-a transgene. The models include=20 platelet-derived growth factor B (PDGFB)-induced = oligodendrogliomas in=20 wild-type mice and Kras-induced spindle gliomas and GBMs in=20 arf-null mice [29,30]. Of these tumors, the Ras and Akt = pathways are=20 upregulated only in Kras-induced tumors and not in PDGFB=20 oligodendrogliomas [31]. Thus, the Kras-induced model more closely = resembles=20 human GBMs and has the potential to exhibit similar translation = effects.=20 To investigate the expression of Notch pathway components, in=20 situ and Western blot analyses were performed on these = different=20 glioma subtypes.
It has been previously reported = that Notch1 is=20 expressed in selected cells within the cerebellum in adult rat = brain,=20 specially in the Purkinje cell layer [34]. We have confirmed that we could obtain a = similar=20 pattern using our antisense probe and had little background with = the=20 corresponding sense probe control (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 23D"Figure,=20 A and B). We analyzed tumors driven from Ntv-a = mice=20 where the cell of origin was nestin-expressing. On in = situ=20 hybridization analysis, Notch1 mRNA is overexpressed in both Ntv-a = PDGF=20 and Ntv-a Kras arf-/- tumors compared to normal brain = (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 2C3D"Figure).=20 Because translation may regulate Notch1 expression, defining = relative mRNA=20 and protein levels may be important in understanding Notch = expression=20 levels. To assess potential translation effect, we = semiquantitatively=20 determined relative mRNA expression levels by comparing in = situ=20 signal density in tumor regions to control normal brain regions = within the=20 same section (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 3A3D"Figure).=20 Images were processed by Metamorph software to determine the total = positive signal image area in each image. Tumor regions were then = compared=20 to normal brain regions from the same slide. Kras-induced tumors = had a=20 5.9-fold higher level of Notch1 signal compared to controls, = whereas=20 PDGFB-induced oligodendrogliomas had an 8.9-fold higher level (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 3B3D"Figure).=20 Expression of Notch1 protein was also found to be higher in both = tumor=20 types compared to normal brain (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 3C3D"Figure).=20 However, Notch1 protein levels were much higher in Kras GBMs = compared to=20 oligodendrogliomas even though in situ analysis revealed = that=20 mRNA levels were relatively lower in Kras GBMs than in PDGF=20 oligodendrogliomas compared to normal brain (5.9-fold vs=20 8.9-fold). This supports our previous finding that Ras and Akt = signaling=20 pathways may selectively recruit Notch1 mRNA into polyribosomes to = be=20 translated into protein [6]. Substantially more active Akt and Ras signaling = pathways=20 are found in Kras-induced GBMs compared to PDGFB-induced=20 oligodendrogliomas and may explain why Notch1 protein levels are = higher in=20 our mouse models of these tumors.
3D"Figure
<= STRONG>Figure=20 1
Notch = receptor and=20 ligand expression in human gliomas. (A) Analysis of Jagged1 = mRNA=20 expression in glioblastomas (GBMs) and non-GBM gliomas = (NGGs)=20 compared to normal brain. Data were derived from the = microarray of=20 Tanwar et al. [32]. (B) Western blot analysis = (more ...)
3D"Figure
<= STRONG>Figure=20 2
Notch = receptor and=20 ligand expression in mouse glioma models. (A) Dark-field = image of=20 antisense Notch1 in situ hybridization, and = hematoxylin and=20 eosin (H&E) image of the same section in the cerebellum. = Positive signal corresponds to a previously = (more ...)
Delta-like1 and Jagged1, = ligands of=20 Notch receptors, were found to be overexpressed by both in = situ=20 and Western blot analyses in Kras-induced tumors (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figures = 2C3D"Figure=20 and <= SPAN=20 style=3D"POSITION: relative; TEXT-DECORATION: none">=E2=80=8Band3C).3C3D"Figure).=20 In PDGFB-induced oligodendrogliomas, these ligands are either not = elevated=20 or only mildly elevated (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figures = 2C3D"Figure=20 and <= SPAN=20 style=3D"POSITION: relative; TEXT-DECORATION: none">=E2=80=8Band3C).3C3D"Figure).=20 One common transcriptional target of Notch signaling is HES1. We = tested=20 for HES1 expression in our mouse tumor models and found it to be=20 overexpressed in situ in Kras arf-/-GBMs = compared to=20 normal tissues (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 2C3D"Figure).=20 In PDGFB oligodendrogliomas, HES1 expression was found to be = comparable to=20 normal tissue or, perhaps, to be slightly elevated on a = cell-to-cell=20 basis. These results suggest that, in Kras-induced GBMs, the Notch = pathway=20 is active because the receptor, ligand, and target are all = expressed. In=20 PDGFB-induced oligodendrogliomas, the receptor appears to be=20 overexpressed; however, of the ligands and targets we examined, = only=20 moderate levels were detected. There may be some degree of Notch = signaling=20 through HES1 in oligodendrogliomas, but the level of activation is = much=20 less compared to that in Kras-induced tumors.
Notch Activation of Target = Genes in=20 Kras-Induced Tumors
Notch acts as a = transcriptional=20 activator by complexing with the DNA-binding protein CBF-1, = displacing=20 repressive factors and recruiting coactivators. Because one effect = of=20 Notch signaling is activation of transcription, an expression = array=20 analysis may reveal its function in Kras-induced tumors. We = compared the=20 expression profile of two Kras-induced tumors to the normal cortex = from=20 Ntv-a arf-/- mice (Table 1 and Table W1). The list of genes upregulated contained = a wide=20 range of proteins involved in various processes. Included in this = list=20 were some expected targets in Kras gliomagenesis. For example, = glial=20 progenitor markers, including vimentin, PDGFR=CE=B1, and nestin, = were=20 increased. Ras signaling components and matrix-degrading enzymes = were also=20 increased. Of 828 genes upregulated by > 3-fold (P = < .05),=20 we searched for CBF-1 consensus sites (YGTGGGAA) in the 5-kb = 5=E2=80=B2 upstream=20 promoter sequence in front of the transcriptional start site using = the=20 annotation provided on http://www.ensembl.org/.=20 Using this search, 136 genes were found to have this motif and to = be=20 potential Notch target genes by binding to CBF-1 (Table 1 and Table W1). Among this list of genes are ones that = have been=20 implicated by other studies as potential targets of Notch = signaling,=20 including cyclin D1 [35], S100a10 [36], and a Snail family member [37]. Not identified by this method but upregulated = on the=20 array and a potential Notch target is the cyclin-dependent kinase=20 inhibitor p21 [38].
3D"Figure
<= STRONG>Figure=20 3
Semiquantitative=20 analysis of Notch1 mRNA and protein in PDGFB and Kras = tumors. (A)=20 Sample dark-field images of Notch1 in situ in = normal and=20 tumor regions of PDGFB and Kras tumors. (B) Image analysis = of=20 positive signal regions in tumors. Positive pixel = (more ...)
One interesting target found by = this search is=20 nestin, an intermediate filament protein that acts as a marker for = neural=20 progenitor cells. Although the 5=E2=80=B2 upstream promoter plays = a role in=20 modulating its expression, it is the nestin second intron that = directs=20 expression in neural precursors [27,39]. Conserved near the 3=E2=80=B2 end of = the second intron=20 of both human and mouse nestins is a CBF-1-binding site (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 4A3D"Figure).=20 We tested whether Notch activation could activate transcription = through=20 this element. The human nestin second intron enhancer element = linked to a=20 tk minimal promoter is sufficient to replicate the neural = expression=20 pattern for nestin [28]. This enhancer-promoter combination was used = to drive=20 the expression of two reporter genes =CE=B2-galactosidase = and=20 luciferase. Nestin reporters were tested in U251 glioma = cell=20 lines in the presence and in the absence of NICD expression. This = form of=20 Notch is equivalent to the proteolytically processed receptor, a=20 constitutively active isoform [8]. We found that NICD could activate the promoter = using=20 either reporter. In the =CE=B2-galactosidase assay, U251 cells = cotransfected=20 with NICD turned blue, whereas those cotransfected with empty = vector did=20 not (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 43D"Figure,=20 B and C). NICD also activated the luciferase = reporter=20 4.8-fold compared to controls (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 4D3D"Figure).=20 These data suggest that Notch may act directly to activate nestin=20 expression in progenitor cells or glioma cells.
3D"Table<= /DIV>
Table=20 1
Selected = List of=20 Genes Upregulated By > 3-Fold in Kras arf-/- = Tumors=20 Compared to arf-/- Cortex.
To assess whether = activation of the=20 Notch pathway correlated with nestin expression in vivo, = we=20 stained Ntv-a Kras arf-/- tumors and Ntv-a PDGFB tumors = for=20 nestin expression. In Kras-induced spindle-like tumors where the = Notch=20 pathway is active, nestin is expressed throughout the tumor (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 4E3D"Figure).=20 However, in PDGFB-induced oligodendrogliomas where HES1, Delta1, = and=20 Jagged1 are not elevated to the same degree, nestin is not clearly = expressed by tumor cells (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 4F3D"Figure).=20 This shows that in vivo nestin expression correlates with = activation of the Notch pathway.
Notch1 Activation Can Cooperate = with Kras=20 to Form Progenitor-Like Lesions
To establish the effect = of Notch1=20 activation on glial tumors, we subcloned a myc-tagged mouse NICD = isoform=20 into the RCAS(A) retroviral system. The construct was tested for=20 functionality by the ability to activate the Hes1 promoter (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5A3D"Figure).=20 To determine whether the expression of NICD in nestin-positive = glial=20 progenitors would be sufficient to induce tumorigenesis, we = infected 39=20 Ntv-a mice with either RCAS NICD virus or RCAS NICD virus with an = empty=20 RCAS virus on postnatal day 1 and observed them for symptoms. We = analyzed=20 21 mice up to 12 weeks and 18 mice up to 24 weeks. None of these = developed=20 any signs of tumor, and histologic analysis of the brains yielded=20 normal-appearing results (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5B3D"Figure).=20 To determine whether Notch signaling could cooperate with other = oncogenes,=20 we coinfected NICD virus with a virus carrying either activated = Akt or=20 Kras=E2=80=94oncogenes previously identified to contribute to = glial tumorigenesis=20 [25]. Furthermore, the delivery of these oncogenes = alone in a=20 wild-type background is insufficient to generate tumors or lesions = in this=20 mouse model [25]. The combination of NICD and Akt failed to = generate any=20 distinguishing abnormalities in 25 tested mice analyzed up to 24 = weeks.=20 However, the combination of NICD and Kras produced periventricular = lesions=20 in 9 of 90 infected mice. Twenty-nine mice were analyzed up to 12 = weeks,=20 and 61 mice were analyzed up to 24 weeks. Regardless of the age of = the=20 mice in which they were found, lesions remained small and limited = to the=20 SVZ without progressing to tumors or larger lesions (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5C3D"Figure).=20 This incidence of lesion formation was statistically significant=20 (P < .05) when compared to mice infected with RCAS = NICD virus=20 alone or combined with a control RCAS virus. These lesions were = then=20 analyzed for immunohistochemical markers (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5D3D"Figure).=20 They are positive for the myc tag on the NICD, indicating = effective viral=20 delivery, and are also positive for nestin and PCNA, indicating = that cells=20 were in active cell cycles. This result confirms in vivo = the=20 potential for nestin to be a direct Notch transcriptional target. = This=20 also suggests that Notch activation can cooperate with Kras to = produce=20 lesions that retain stem-like character by their location in the = SVZ,=20 continued proliferation, and progenitor marker expression. Nestin=20 expression is maintained either through Notch's direct action on = its=20 promoter or through Notch's effect on maintaining an = undifferentiated=20 state in nestin-positive progenitors.
3D"Figure
<= STRONG>Figure=20 4
Notch1 = activation=20 of the nestin second intron element. (A) Alignment of human = and=20 mouse Nes second intron segments. Boxed regions indicate = potential=20 CBF-1-binding site. Numbering based on GenBank sequences: = human=20 AF004335=20 and mouse AY438043.=20 (B and C) = (more ...)
Ntv-a mice support gene = transfer to=20 nestin-expressing cells; in this case, Notch activity may simply = maintain=20 nestin expression. We next used Gtv-a mice to determine the = ability of=20 Notch activation to drive nestin expression in tumors derived from = a=20 GFAP-expressing cell of origin, a more differentiated cell type = that has=20 already lost nestin expression. We cloned an HA-tagged version of = rat=20 Delta1 (a ligand for Notch receptor) into the RCAS vector and = assessed its=20 expression using Western blot detection of the HA tag in Ntv-a=20 mouse-derived glial cells (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5E3D"Figure).=20 We then infected Gtv-a arf-/- mice with RCAS-Kras,=20 RCAS-rDelta1-HA, or a combination of the two. Gtv-a = arf-/- mice=20 express the viral-targeting receptor on GFAP-positive cells, and=20 RCAS-mediated Kras oncogene delivery to these cells induces a = similar=20 spindle tumor with moderate nestin expression, as seen with Ntv-a=20 arf-/- mice [29]. These tumors have abundant Notch1 expression, = as=20 determined by in situ analysis. Expression of rDelta1-HA = alone in=20 these mice was insufficient to induce tumors. Mice infected with a = combination of RCAS-Kras and RCAS-rDelta1-HA developed tumors and = were=20 sacrificed when symptomatic or at 12 weeks. These tumors were then = analyzed for HA and nestin expression. HA-staining regions are = composed of=20 Kras-driven tumors that have additional Notch activation by the = expression=20 of rDelta1. In analyzing the five large tumors that developed, in = regions=20 where Kras alone was expressed (with no HA staining), nestin = expression=20 was intermediate throughout (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5F3D"Figure).=20 However, nestin expression was markedly elevated where rDelta1 was = coexpressed (as determined with positive HA staining) with Kras = (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5F3D"Figure).=20 This demonstrates that elevated Notch signaling upregulates nestin = expression in glial tumors, even from a GFAP-expressing cell of = origin,=20 and can collaborate with Kras signaling to alter tumor=20 phenotype.
Discussion
Our study demonstrates = that Notch=20 signaling can play important roles in glial tumor development,=20 particularly in promoting nestin expression that may contribute to = stem=20 cell potential. We were able to identify Notch receptors and = ligands in=20 human glial tumors, suggesting that juxtacrine or autocrine modes = of=20 activation are possible=E2=80=94a situation that most often occurs = in GBMs, the=20 highest grade of glial tumors. Previous results have indicated = higher=20 Notch1 mRNA in grade II to grade III astrocytomas than in GBMs, no = significant elevation of Delta-like1 mRNA in astrocytomas and = GBMs, and=20 elevation of Jagged1 in only a subset of GBMs [23]. Our results agree with this previous report = on the=20 elevated level of Jagged1 in a subset of GBMs. However, at the = protein=20 level, we observed similar levels of Notch1 receptor in human = glial tumors=20 and higher ligand levels in GBMs. The lack of correlation between = mRNA and=20 protein levels is consistent with previous reports showing that = Notch1 is=20 one mRNA that is translationally regulated through its recruitment = into=20 polyribosomes by signaling pathways known to be active in GBMs [6]. In this study, we observed a similar finding in = mouse=20 tumors with elevated Notch1 protein in Kras-induced GBMs, where = Ras and=20 Akt pathways are significantly elevated. Thus, equivalent mRNA = levels may=20 lead to differing amounts of protein. We have also identified = Notch2=20 protein to be expressed in selected GBMs. Thus, our results add to = the=20 growing literature supporting the importance of Notch signaling in = the=20 formation of human GBMs.
In our mouse glial tumor models, = we found=20 that, in Krasinduced GBMs, Notch1 receptor, Delta-like1, and = Jagged1 are=20 upregulated, accompanied by increased transcription of HES1, a = common=20 Notch transcriptional target. The cooperative nature of activated = Notch=20 and Kras signaling was additionally observed with the ability of = these two=20 genes combined to generate lesions located in the SVZ, the = location of=20 neural stem cells. The cells in these lesions continue to express = a=20 proliferation marker and also nestin, much as stem-like cells do. = The=20 ability of Notch to sustain the expression of the progenitor = marker nestin=20 may have a direct effect on transcription by Notch at the nestin = second=20 intron enhancer element. Based on these findings, Notch activation = appears=20 capable of promoting or sustaining nestin expression and the = stem-like=20 character of SVZ cells.
Given the diverse roles that the = Notch pathway=20 plays in normal glial development, parallels between development = and tumor=20 formation may be seen. As in development, persistent Notch = activation may=20 be a means to keep cells in a more undifferentiated progenitor = state. It=20 is believed that cancer stem cells may be the source of tumor = cells and=20 that SVZ is thought to be the origin of neural stem cells in the = cerebrum=20 [40]. The periventricular lesions seen with = combined Notch=20 and Kras infections in wild-type mice may be a reflection of this = aspect=20 of Notch's function. In normal development, nestin-positive cells = migrate=20 from the SVZ into the brain on postnatal day 0 and are limited to = a small=20 zone of periventricular cells in the adult brain [41]. By activating the Notch and Ras pathways in=20 nestin-positive progenitors, these cells appear capable of = continued=20 proliferation in adult mice in the SVZ as if they have been = prevented from=20 proceeding through a normal differentiation pathway.
In Kras-generated GBMs, the = activation of the=20 MAP kinase Erk and Akt pathways may affect protein translation. = Thus,=20 compared to PDGFB-generated oligodendrogliomas, Kras-induced GBMs = produce=20 more Notch proteins from relatively similar amounts of Notch mRNA. = The=20 expression of Notch ligands Delta1 and Jagged1 appears to be = regulated by=20 transcriptional mechanisms that upregulate expression in Kras = tumors and=20 not in PDGFB tumors. The coexpression of ligand and receptor in=20 Kras-induced GBMs may be responsible for juxtacrine Notch = signaling that=20 can maintain progenitor characteristics. Therefore, nestin = expression is=20 correlated with Kras-induced GBM-like tumors and not with = PDGFB-induced=20 oligodendrogliomas. In PDGFB-generated oligodendrogliomas, the = Notch1=20 receptor is overexpressed, but the ligands and Hes1 target are = either=20 minimally expressed or at levels slightly higher than that in = normal=20 brain. This, however, does not exclude Notch function from these = tumors.=20 There are three Delta-like isoforms and two Jagged isoforms, in = addition=20 to a newly identified ligand F-contactin that has been shown to = have=20 specific effects on oligodendrocytes [42]. Alternatively, the lower level of receptor = and ligand=20 expression seen in Western blot analysis may still be sufficient = for=20 signaling.
Inhibition of Notch = activity may be=20 useful for glioma therapy. One possible target is presenilin = =CE=B3 secretase,=20 which acts to cleave and release Notch from membranes. The = combination of=20 a Notch inhibitor and Ras inhibitor may be particularly effective = because=20 these pathways seem to work synergistically to induce lesions and = to=20 sustain Ras effects. Further studies that elucidate the molecular=20 mechanisms and specific targets of Notch signaling, particularly = ones that=20 distinguish its multiple functions, will also bring greater = insight into=20 glioma biology.
Supplementary=20 Material
Supplementary Figures and = Tables
Click=20 here to view.(19K, pdf)
Acknowledgements
We thank Hiroyuki Momota for = critical=20 suggestions on the manuscript; Edward Nerio and Jim Finney for = technical=20 assistance; Veronique Bourdon for microarray analysis; and David = Anderson,=20 Gerry Weinmaster, and Urban Lendahl for the=20 reagents.
Footnotes
1Laboratory work was = supported by=20 the Kleeberg Foundation, Kirby Foundation, and National Institutes = of=20 Health (NIH) grants UO1CA894314-1 and RO1 CA099489=20 to E.C.H. A.H.S. was supported by NIH MSTP grant GM07739.
*This article refers = to=20 supplementary material, which is designated by =E2=80=9CW=E2=80=9D = (i.e., Table W1) and is available online at http://www.bcdecker.com/.
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Articles from Neoplasia (New=20 York, N.Y.) are provided here courtesy of
Neoplasia Press
3D"Figure
<= STRONG>Figure=20 5
Activated = Notch and=20 Kras can induce lesions in Ntv-a-targeted mice. (A) HES1 = promoter=20 luciferase assay with empty plasmid control or RCAS NICD=20 transfection. (B) Table of lesion incidence in NICD/NICD + = control=20 virus, NICD + Akt, and NICD + Kras infections = (more ...)

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  • Shih,=20 A.
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.x-panel-nofooter .x-window-bc { FONT-SIZE: 0px; LINE-HEIGHT: 0; HEIGHT: 6px } .x-panel-bwrap { LEFT: 0px; OVERFLOW: hidden; ZOOM: 1; TOP: 0px } .x-panel-body { OVERFLOW: hidden; ZOOM: 1 } .x-panel-collapsed .x-resizable-handle { DISPLAY: none } .ext-gecko .x-panel-animated DIV { OVERFLOW: hidden! important } .x-plain-body { OVERFLOW: hidden } .x-plain-bbar .x-toolbar { PADDING-RIGHT: 2px; PADDING-LEFT: 2px; PADDING-BOTTOM: 2px; OVERFLOW: = hidden; PADDING-TOP: 2px } .x-plain-tbar .x-toolbar { PADDING-RIGHT: 2px; PADDING-LEFT: 2px; PADDING-BOTTOM: 2px; OVERFLOW: = hidden; PADDING-TOP: 2px } .x-plain-bwrap { OVERFLOW: hidden; ZOOM: 1 } .x-plain { OVERFLOW: hidden } .x-tool { BACKGROUND: url(../images/default/panel/tool-sprites.gif) no-repeat; = FLOAT: right; MARGIN-LEFT: 2px; OVERFLOW: hidden; WIDTH: 15px; CURSOR: = pointer; HEIGHT: 15px } .x-tool-toggle { BACKGROUND-POSITION: 0px -60px } .x-tool-toggle-over { BACKGROUND-POSITION: -15px -60px } .x-panel-collapsed .x-tool-toggle { BACKGROUND-POSITION: 0px -75px } .x-panel-collapsed .x-tool-toggle-over { BACKGROUND-POSITION: -15px -75px } .x-tool-close { BACKGROUND-POSITION: 0px 0px } .x-tool-close-over { BACKGROUND-POSITION: -15px 0px } .x-tool-minimize { BACKGROUND-POSITION: 0px -15px } .x-tool-minimize-over { BACKGROUND-POSITION: -15px -15px } .x-tool-maximize { BACKGROUND-POSITION: 0px -30px } .x-tool-maximize-over { BACKGROUND-POSITION: -15px -30px } .x-tool-restore { BACKGROUND-POSITION: 0px -45px } .x-tool-restore-over { BACKGROUND-POSITION: -15px -45px } .x-tool-gear { BACKGROUND-POSITION: 0px -90px } .x-tool-gear-over { BACKGROUND-POSITION: -15px -90px } .x-tool-pin { BACKGROUND-POSITION: 0px -135px } .x-tool-pin-over { BACKGROUND-POSITION: -15px -135px } .x-tool-unpin { BACKGROUND-POSITION: 0px -150px } .x-tool-unpin-over { BACKGROUND-POSITION: -15px -150px } .x-tool-right { BACKGROUND-POSITION: 0px -165px } .x-tool-right-over { BACKGROUND-POSITION: -15px -165px } .x-tool-left { BACKGROUND-POSITION: 0px -180px } .x-tool-left-over { BACKGROUND-POSITION: -15px -180px } .x-tool-up { BACKGROUND-POSITION: 0px -210px } .x-tool-up-over { BACKGROUND-POSITION: -15px -210px } .x-tool-down { BACKGROUND-POSITION: 0px -195px } .x-tool-down-over { BACKGROUND-POSITION: -15px -195px } .x-tool-refresh { BACKGROUND-POSITION: 0px -225px } .x-tool-refresh-over { BACKGROUND-POSITION: -15px -225px } .x-tool-minus { BACKGROUND-POSITION: 0px -255px } .x-tool-minus-over { BACKGROUND-POSITION: -15px -255px } .x-tool-plus { BACKGROUND-POSITION: 0px -240px } .x-tool-plus-over { BACKGROUND-POSITION: -15px -240px } .x-tool-search { BACKGROUND-POSITION: 0px -270px } .x-tool-search-over { BACKGROUND-POSITION: -15px -270px } .x-tool-save { BACKGROUND-POSITION: 0px -285px } .x-tool-save-over { BACKGROUND-POSITION: -15px -285px } .x-tool-help { BACKGROUND-POSITION: 0px -300px } .x-tool-help-over { BACKGROUND-POSITION: -15px -300px } .x-tool-print { BACKGROUND-POSITION: 0px -315px } .x-tool-print-over { BACKGROUND-POSITION: -15px -315px } .x-panel-ghost { Z-INDEX: 12000; BACKGROUND: #cbddf3; FILTER: alpha(opacity=3D65); LEFT: = 0px; OVERFLOW: hidden; POSITION: absolute; TOP: 0px; opacity: .65; = -moz-opacity: .65 } .x-panel-ghost UL { BORDER-RIGHT: #99bbe8 1px solid; PADDING-RIGHT: 0px; BORDER-TOP: = #99bbe8 0px; DISPLAY: block; PADDING-LEFT: 0px; FONT-SIZE: 0px; = PADDING-BOTTOM: 0px; MARGIN: 0px; OVERFLOW: hidden; BORDER-LEFT: #99bbe8 = 1px solid; LINE-HEIGHT: 0; PADDING-TOP: 0px; BORDER-BOTTOM: #99bbe8 1px = solid } .x-panel-ghost * { CURSOR: move! important } .x-panel-dd-spacer { BORDER-RIGHT: #99bbe8 2px dashed; BORDER-TOP: #99bbe8 2px dashed; = BORDER-LEFT: #99bbe8 2px dashed; BORDER-BOTTOM: #99bbe8 2px dashed } .x-panel-btns-ct { PADDING-RIGHT: 5px; PADDING-LEFT: 5px; PADDING-BOTTOM: 5px; = PADDING-TOP: 5px } .x-panel-btns-ct .x-btn { CLEAR: none; FLOAT: right } .x-panel-btns-ct .x-panel-btns TD { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = PADDING-TOP: 0px; BORDER-RIGHT-WIDTH: 0px } .x-panel-btns-ct .x-panel-btns-right TABLE { CLEAR: none; FLOAT: right } .x-panel-btns-ct .x-panel-btns-left TABLE { CLEAR: none; FLOAT: left } .x-panel-btns-ct .x-panel-btns-center { TEXT-ALIGN: center } .x-panel-btns-ct .x-panel-btns-center TABLE { MARGIN: 0px auto } .x-panel-btns-ct TABLE TD.x-panel-btn-td { PADDING-RIGHT: 3px; PADDING-LEFT: 3px; PADDING-BOTTOM: 3px; = PADDING-TOP: 3px } .x-panel-btns-ct .x-btn-focus .x-btn-left { BACKGROUND-POSITION: 0px -147px } .x-panel-btns-ct .x-btn-focus .x-btn-right { BACKGROUND-POSITION: 0px -168px } .x-panel-btns-ct .x-btn-focus .x-btn-center { BACKGROUND-POSITION: 0px -189px } .x-panel-btns-ct .x-btn-over .x-btn-left { BACKGROUND-POSITION: 0px -63px } .x-panel-btns-ct .x-btn-over .x-btn-right { BACKGROUND-POSITION: 0px -84px } .x-panel-btns-ct .x-btn-over .x-btn-center { BACKGROUND-POSITION: 0px -105px } .x-panel-btns-ct .x-btn-click .x-btn-center { BACKGROUND-POSITION: 0px -126px } .x-panel-btns-ct .x-btn-click .x-btn-right { BACKGROUND-POSITION: 0px -84px } .x-panel-btns-ct .x-btn-click .x-btn-left { BACKGROUND-POSITION: 0px -63px } ------=_NextPart_000_0000_01CAD4DC.9F0D8F00 Content-Type: text/css; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable Content-Location: http://www.ncbi.nlm.nih.gov/core/ext.ncbi/1/css/themes/xtheme-oldentrez.css .x-panel { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-STYLE: solid; BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-STYLE: = solid; BORDER-LEFT-STYLE: solid; BORDER-RIGHT-COLOR: #d0d0d0; = BORDER-BOTTOM-STYLE: solid } .x-panel-header { BORDER-RIGHT: #d0d0d0 1px solid; BORDER-TOP: #d0d0d0 1px solid; = BACKGROUND-IMAGE: url(../../img/oldentrez/panel/white-top-bottom.gif); = BORDER-LEFT: #d0d0d0 1px solid; COLOR: #333; BORDER-BOTTOM: #d0d0d0 1px = solid } .x-panel-body { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-bbar .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-tbar .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-tbar-noheader .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-mc .x-panel-tbar .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-body-noheader { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-mc .x-panel-body { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-tl .x-panel-header { COLOR: #333 } .x-panel-tc { BACKGROUND-IMAGE: url(../../img/oldentrez/panel/top-bottom.gif) } .x-panel-tl { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND-IMAGE: = url(../../img/oldentrez/panel/corners-sprite.gif); BORDER-BOTTOM-COLOR: = #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-tr { BACKGROUND-IMAGE: url(../../img/oldentrez/panel/corners-sprite.gif) } .x-panel-bc { BACKGROUND-IMAGE: url(../../img/oldentrez/panel/top-bottom.gif) } .x-panel-bl { BACKGROUND-IMAGE: url(../../img/oldentrez/panel/corners-sprite.gif) } .x-panel-br { BACKGROUND-IMAGE: url(../../img/oldentrez/panel/corners-sprite.gif) } .x-panel-mc { BACKGROUND: #f1f1f1 } .x-panel-mc .x-panel-body { BORDER-RIGHT: 0px; BORDER-TOP: 0px; BACKGROUND: none transparent scroll = repeat 0% 0%; BORDER-LEFT: 0px; BORDER-BOTTOM: 0px } .x-panel-ml { BACKGROUND-IMAGE: url(../../img/oldentrez/panel/left-right.gif) } .x-panel-mr { BACKGROUND-IMAGE: url(../../img/oldentrez/panel/left-right.gif) } .x-tool { BACKGROUND-IMAGE: url(../../img/oldentrez/panel/tool-sprites.gif) } .x-panel-ghost { BACKGROUND: #e0e0e0 } .x-panel-ghost UL { BORDER-LEFT-COLOR: #b0b0b0; BORDER-BOTTOM-COLOR: #b0b0b0; = BORDER-TOP-COLOR: #b0b0b0; BORDER-RIGHT-COLOR: #b0b0b0 } .x-grid-panel .x-panel-mc .x-panel-body { BORDER-RIGHT: #d0d0d0 1px solid; BORDER-TOP: #d0d0d0 1px solid; = BORDER-LEFT: #d0d0d0 1px solid; BORDER-BOTTOM: #d0d0d0 1px solid } .x-btn-left { BACKGROUND-IMAGE: url(../../img/oldentrez/button/btn-sprite.gif) } .x-btn-right { BACKGROUND-IMAGE: url(../../img/oldentrez/button/btn-sprite.gif) } .x-btn-center { BACKGROUND-IMAGE: url(../../img/oldentrez/button/btn-sprite.gif) } .x-border-layout-ct { BACKGROUND: #f0f0f0 } .x-accordion-hd { BACKGROUND-IMAGE: url(../../img/oldentrez/panel/light-hd.gif) } .x-layout-collapsed { BORDER-LEFT-COLOR: #e0e0e0; BORDER-BOTTOM-COLOR: #e0e0e0; = BORDER-TOP-COLOR: #e0e0e0; BACKGROUND-COLOR: #eee; BORDER-RIGHT-COLOR: = #e0e0e0 } .x-layout-collapsed-over { BACKGROUND-COLOR: #fbfbfb } .x-tip .x-tip-top { BACKGROUND-IMAGE: url(../../img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-top-left { BACKGROUND-IMAGE: url(../../img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-top-right { BACKGROUND-IMAGE: url(../../img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-ft { BACKGROUND-IMAGE: url(../../img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-ft-left { BACKGROUND-IMAGE: url(../../img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-ft-right { BACKGROUND-IMAGE: url(../../img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-bd-left { BACKGROUND-IMAGE: url(../../img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-bd-right { BACKGROUND-IMAGE: url(../../img/oldentrez/qtip/tip-sprite.gif) } .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND: = url(../../img/oldentrez/toolbar/bg.gif) #f0f4f5 repeat-x left top; = BORDER-BOTTOM-COLOR: #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; = BORDER-RIGHT-COLOR: #d0d0d0 } .x-toolbar BUTTON { COLOR: #444 } .x-toolbar .x-btn-menu-arrow-wrap .x-btn-center BUTTON { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/btn-arrow.gif) } .x-toolbar .x-btn-text-icon .x-btn-menu-arrow-wrap .x-btn-center BUTTON = { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/btn-arrow.gif) } .x-toolbar .x-btn-over .x-btn-left { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-over .x-btn-right { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-over .x-btn-center { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-over BUTTON { COLOR: #111 } .x-toolbar .x-btn-click .x-btn-left { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-pressed .x-btn-left { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-menu-active .x-btn-left { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-click .x-btn-right { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-pressed .x-btn-right { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-menu-active .x-btn-right { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-click .x-btn-center { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-pressed .x-btn-center { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-menu-active .x-btn-center { BACKGROUND-IMAGE: url(../../img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .ytb-sep { BACKGROUND-IMAGE: url(../images/default/grid/grid-split.gif) } .x-tab-panel-header { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND: #eaeaea; BORDER-BOTTOM-COLOR: = #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-panel-footer { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND: #eaeaea; BORDER-BOTTOM-COLOR: = #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-panel-header { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-panel-footer { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } UL.x-tab-strip-top { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND: = url(../../img/oldentrez/tabs/tab-strip-bg.gif) #dbdbdb repeat-x left = top; BORDER-BOTTOM-COLOR: #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; = PADDING-TOP: 2px; BORDER-RIGHT-COLOR: #d0d0d0 } UL.x-tab-strip-bottom { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND-IMAGE: = url(../../img/oldentrez/tabs/tab-strip-btm-bg.gif); BORDER-BOTTOM-COLOR: = #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-strip SPAN.x-tab-strip-text { COLOR: #333 } .x-tab-strip-over SPAN.x-tab-strip-text { COLOR: #111 } .x-tab-strip-active SPAN.x-tab-strip-text { COLOR: #333 } .x-tab-strip-disabled .x-tabs-text { COLOR: #aaaaaa } .x-tab-strip-top .x-tab-right { BACKGROUND-IMAGE: url(../../img/oldentrez/tabs/tabs-sprite.gif) } .x-tab-strip-top .x-tab-left { BACKGROUND-IMAGE: url(../../img/oldentrez/tabs/tabs-sprite.gif) } .x-tab-strip-top .x-tab-strip-inner { BACKGROUND-IMAGE: url(../../img/oldentrez/tabs/tabs-sprite.gif) } .x-tab-strip-bottom .x-tab-right { BACKGROUND-IMAGE: = url(../../img/oldentrez/tabs/tab-btm-inactive-right-bg.gif) } .x-tab-strip-bottom .x-tab-left { BACKGROUND-IMAGE: = url(../../img/oldentrez/tabs/tab-btm-inactive-left-bg.gif) } .x-tab-strip-bottom .x-tab-strip-active .x-tab-right { BACKGROUND-IMAGE: url(../../img/oldentrez/tabs/tab-btm-right-bg.gif) } .x-tab-strip-bottom .x-tab-strip-active .x-tab-left { BACKGROUND-IMAGE: url(../../img/oldentrez/tabs/tab-btm-left-bg.gif) } .x-tab-strip .x-tab-strip-closable A.x-tab-strip-close { BACKGROUND-IMAGE: url(../../img/oldentrez/tabs/tab-close.gif) } .x-tab-strip .x-tab-strip-closable A.x-tab-strip-close:hover { BACKGROUND-IMAGE: url(../../img/oldentrez/tabs/tab-close.gif) } .x-tab-panel-body { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND: #fff; BORDER-BOTTOM-COLOR: = #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-panel-bbar .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-panel-tbar .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-panel-header-plain .x-tab-strip-spacer { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND: #eaeaea; BORDER-BOTTOM-COLOR: = #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-scroller-left { BORDER-LEFT-COLOR: #aeaeae; BACKGROUND-IMAGE: = url(../../img/oldentrez/tabs/scroll-left.gif); BORDER-BOTTOM-COLOR: = #aeaeae; BORDER-TOP-COLOR: #aeaeae; BORDER-RIGHT-COLOR: #aeaeae } .x-tab-scroller-right { BORDER-LEFT-COLOR: #aeaeae; BACKGROUND-IMAGE: = 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.x-panel-footer { PADDING-BOTTOM: 6px } .x-panel-nofooter .x-panel-bc { FONT-SIZE: 0px; LINE-HEIGHT: 0; HEIGHT: 6px } .x-panel-nofooter .x-window-bc { FONT-SIZE: 0px; LINE-HEIGHT: 0; HEIGHT: 6px } .x-panel-bwrap { OVERFLOW: hidden; ZOOM: 1 } .x-panel-body { OVERFLOW: hidden; ZOOM: 1 } .x-panel-collapsed .x-resizable-handle { DISPLAY: none } .ext-gecko .x-panel-animated DIV { OVERFLOW: hidden! important } .x-plain-body { OVERFLOW: hidden } .x-plain-bbar .x-toolbar { PADDING-RIGHT: 2px; PADDING-LEFT: 2px; PADDING-BOTTOM: 2px; OVERFLOW: = hidden; PADDING-TOP: 2px } .x-plain-tbar .x-toolbar { PADDING-RIGHT: 2px; PADDING-LEFT: 2px; PADDING-BOTTOM: 2px; OVERFLOW: = hidden; PADDING-TOP: 2px } .x-plain-bwrap { OVERFLOW: hidden; ZOOM: 1 } .x-plain { OVERFLOW: hidden } .x-tool { BACKGROUND: = url(/core/extjs/ext-2.1/resources/images/default/panel/tool-sprites.gif) = no-repeat; FLOAT: right; MARGIN-LEFT: 2px; OVERFLOW: hidden; WIDTH: = 15px; CURSOR: pointer; HEIGHT: 15px } .x-tool-toggle { BACKGROUND-POSITION: 0px -60px } .x-tool-toggle-over { BACKGROUND-POSITION: -15px -60px } .x-panel-collapsed .x-tool-toggle { BACKGROUND-POSITION: 0px -75px } .x-panel-collapsed .x-tool-toggle-over { BACKGROUND-POSITION: -15px -75px } .x-tool-close { BACKGROUND-POSITION: 0px 0px } .x-tool-close-over { BACKGROUND-POSITION: -15px 0px } .x-tool-minimize { BACKGROUND-POSITION: 0px -15px } .x-tool-minimize-over { BACKGROUND-POSITION: -15px 50% } .x-tool-maximize { BACKGROUND-POSITION: 0px -30px } .x-tool-maximize-over { BACKGROUND-POSITION: -15px -30px } .x-tool-restore { BACKGROUND-POSITION: 0px -45px } .x-tool-restore-over { BACKGROUND-POSITION: -15px -45px } .x-tool-gear { BACKGROUND-POSITION: 0px -90px } .x-tool-gear-over { BACKGROUND-POSITION: -15px -90px } .x-tool-pin { BACKGROUND-POSITION: 0px -135px } .x-tool-pin-over { BACKGROUND-POSITION: -15px -135px } .x-tool-unpin { BACKGROUND-POSITION: 0px -150px } .x-tool-unpin-over { BACKGROUND-POSITION: -15px -150px } .x-tool-right { BACKGROUND-POSITION: 0px -165px } .x-tool-right-over { BACKGROUND-POSITION: -15px -165px } .x-tool-left { BACKGROUND-POSITION: 0px -180px } .x-tool-left-over { BACKGROUND-POSITION: -15px -180px } .x-tool-up { BACKGROUND-POSITION: 0px -210px } .x-tool-up-over { BACKGROUND-POSITION: -15px -210px } .x-tool-down { BACKGROUND-POSITION: 0px -195px } .x-tool-down-over { BACKGROUND-POSITION: -15px -195px } .x-tool-refresh { BACKGROUND-POSITION: 0px -225px } .x-tool-refresh-over { BACKGROUND-POSITION: -15px -225px } .x-tool-minus { BACKGROUND-POSITION: 0px -255px } .x-tool-minus-over { BACKGROUND-POSITION: -15px -255px } .x-tool-plus { BACKGROUND-POSITION: 0px -240px } .x-tool-plus-over { BACKGROUND-POSITION: -15px -240px } .x-tool-search { BACKGROUND-POSITION: 0px -270px } .x-tool-search-over { BACKGROUND-POSITION: -15px -270px } .x-tool-save { BACKGROUND-POSITION: 0px -285px } .x-tool-save-over { BACKGROUND-POSITION: -15px -285px } .x-tool-help { BACKGROUND-POSITION: 0px -300px } .x-tool-help-over { BACKGROUND-POSITION: -15px -300px } .x-tool-print { BACKGROUND-POSITION: 0px -315px } .x-tool-print-over { BACKGROUND-POSITION: -15px -315px } .x-panel-ghost { Z-INDEX: 12000; BACKGROUND: #cbddf3; FILTER: alpha(opacity=3D65); LEFT: = 0px; OVERFLOW: hidden; POSITION: absolute; TOP: 0px; opacity: .65; = -moz-opacity: .65 } .x-panel-ghost UL { BORDER-RIGHT: #99bbe8 1px solid; PADDING-RIGHT: 0px; BORDER-TOP: = #99bbe8 0px; DISPLAY: block; PADDING-LEFT: 0px; FONT-SIZE: 0px; = PADDING-BOTTOM: 0px; MARGIN: 0px; OVERFLOW: hidden; BORDER-LEFT: #99bbe8 = 1px solid; LINE-HEIGHT: 0; PADDING-TOP: 0px; BORDER-BOTTOM: #99bbe8 1px = solid } .x-panel-ghost * { CURSOR: move! important } .x-panel-dd-spacer { BORDER-RIGHT: #99bbe8 2px dashed; BORDER-TOP: #99bbe8 2px dashed; = BORDER-LEFT: #99bbe8 2px dashed; BORDER-BOTTOM: #99bbe8 2px dashed } .x-panel-btns-ct { PADDING-RIGHT: 5px; PADDING-LEFT: 5px; PADDING-BOTTOM: 5px; = PADDING-TOP: 5px } .x-panel-btns-ct .x-btn { CLEAR: none; FLOAT: right } .x-panel-btns-ct .x-panel-btns TD { BORDER-TOP-WIDTH: 0px; PADDING-RIGHT: 0px; PADDING-LEFT: 0px; = BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: 0px; PADDING-BOTTOM: 0px; = PADDING-TOP: 0px; BORDER-RIGHT-WIDTH: 0px } .x-panel-btns-ct .x-panel-btns-right TABLE { CLEAR: none; FLOAT: right } .x-panel-btns-ct .x-panel-btns-left TABLE { CLEAR: none; FLOAT: left } .x-panel-btns-ct .x-panel-btns-center { TEXT-ALIGN: center } .x-panel-btns-ct .x-panel-btns-center TABLE { MARGIN: 0px auto } .x-panel-btns-ct TABLE TD.x-panel-btn-td { PADDING-RIGHT: 3px; PADDING-LEFT: 3px; PADDING-BOTTOM: 3px; = PADDING-TOP: 3px } .x-panel-btns-ct .x-btn-focus .x-btn-left { BACKGROUND-POSITION: 0px -147px } .x-panel-btns-ct .x-btn-focus .x-btn-right { BACKGROUND-POSITION: 0px -168px } .x-panel-btns-ct .x-btn-focus .x-btn-center { BACKGROUND-POSITION: 0px -189px } .x-panel-btns-ct .x-btn-over .x-btn-left { BACKGROUND-POSITION: 0px -63px } .x-panel-btns-ct .x-btn-over .x-btn-right { BACKGROUND-POSITION: 0px -84px } .x-panel-btns-ct .x-btn-over .x-btn-center { BACKGROUND-POSITION: 0px -105px } .x-panel-btns-ct .x-btn-click .x-btn-center { BACKGROUND-POSITION: 0px -126px } .x-panel-btns-ct .x-btn-click .x-btn-right { BACKGROUND-POSITION: 0px -84px } .x-panel-btns-ct .x-btn-click .x-btn-left { BACKGROUND-POSITION: 0px -63px } .x-btn-menu-wrap { WIDTH: 0px! important } .x-menu { BACKGROUND: #f0f0f0 0px 0px } .x-menu-list-item IMG { DISPLAY: none } .x-menu-list-item A.x-menu-item { PADDING-LEFT: 8px } .port H1.x-panel-header-text { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; FONT-SIZE: 100%; PADDING-BOTTOM: = 0px; MARGIN: 0px; PADDING-TOP: 0px } .port H2.x-panel-header-text { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; FONT-SIZE: 100%; PADDING-BOTTOM: = 0px; MARGIN: 0px; PADDING-TOP: 0px } .port H3.x-panel-header-text { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; FONT-SIZE: 100%; PADDING-BOTTOM: = 0px; MARGIN: 0px; PADDING-TOP: 0px } .port H4.x-panel-header-text { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; FONT-SIZE: 100%; PADDING-BOTTOM: = 0px; MARGIN: 0px; PADDING-TOP: 0px } .port H5.x-panel-header-text { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; FONT-SIZE: 100%; PADDING-BOTTOM: = 0px; MARGIN: 0px; PADDING-TOP: 0px } .port H6.x-panel-header-text { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; FONT-SIZE: 100%; PADDING-BOTTOM: = 0px; MARGIN: 0px; PADDING-TOP: 0px } DIV.x-panel-body { PADDING-RIGHT: 5px; PADDING-LEFT: 5px; PADDING-BOTTOM: 5px; = PADDING-TOP: 5px } .port .x-panel-header { PADDING-RIGHT: 3px; PADDING-LEFT: 5px; PADDING-BOTTOM: 4px; = PADDING-TOP: 5px } .x-panel { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-STYLE: solid; BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-STYLE: = solid; BORDER-LEFT-STYLE: solid; BORDER-RIGHT-COLOR: #d0d0d0; = BORDER-BOTTOM-STYLE: solid } .x-panel-header { BORDER-RIGHT: #d0d0d0 1px solid; BORDER-TOP: #d0d0d0 1px solid; = BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/panel/white-top-bottom.gif); = BORDER-LEFT: #d0d0d0 1px solid; COLOR: #333; BORDER-BOTTOM: #d0d0d0 1px = solid } .x-panel-body { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-bbar .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-tbar .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-tbar-noheader .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-mc .x-panel-tbar .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-body-noheader { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-mc .x-panel-body { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-tl .x-panel-header { COLOR: #333 } .x-panel-tc { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/panel/top-bottom.gif) } .x-panel-tl { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/panel/corners-sprite.gif); = BORDER-BOTTOM-COLOR: #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; = BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-tr { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/panel/corners-sprite.gif) } .x-panel-bc { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/panel/top-bottom.gif) } .x-panel-bl { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/panel/corners-sprite.gif) } .x-panel-br { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/panel/corners-sprite.gif) } .x-panel-mc { BACKGROUND: #f1f1f1 } .x-panel-mc .x-panel-body { BORDER-RIGHT: 0px; BORDER-TOP: 0px; BACKGROUND: none transparent scroll = repeat 0% 0%; BORDER-LEFT: 0px; BORDER-BOTTOM: 0px } .x-panel-ml { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/panel/left-right.gif) } .x-panel-mr { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/panel/left-right.gif) } .x-tool { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/panel/tool-sprites.gif) } .x-panel-ghost { BACKGROUND: #e0e0e0 } .x-panel-ghost UL { BORDER-LEFT-COLOR: #b0b0b0; BORDER-BOTTOM-COLOR: #b0b0b0; = BORDER-TOP-COLOR: #b0b0b0; BORDER-RIGHT-COLOR: #b0b0b0 } .x-grid-panel .x-panel-mc .x-panel-body { BORDER-RIGHT: #d0d0d0 1px solid; BORDER-TOP: #d0d0d0 1px solid; = BORDER-LEFT: #d0d0d0 1px solid; BORDER-BOTTOM: #d0d0d0 1px solid } .x-btn-left { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/button/btn-sprite.gif) } .x-btn-right { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/button/btn-sprite.gif) } .x-btn-center { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/button/btn-sprite.gif) } .x-border-layout-ct { BACKGROUND: #f0f0f0 } .x-accordion-hd { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/panel/light-hd.gif) } .x-layout-collapsed { BORDER-LEFT-COLOR: #e0e0e0; BORDER-BOTTOM-COLOR: #e0e0e0; = BORDER-TOP-COLOR: #e0e0e0; BACKGROUND-COLOR: #eee; BORDER-RIGHT-COLOR: = #e0e0e0 } .x-layout-collapsed-over { BACKGROUND-COLOR: #fbfbfb } .x-tip .x-tip-top { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-top-left { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-top-right { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-ft { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-ft-left { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-ft-right { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-bd-left { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/qtip/tip-sprite.gif) } .x-tip .x-tip-bd-right { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/qtip/tip-sprite.gif) } .x-toolbar { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/bg.gif) #f0f4f5 repeat-x left = top; BORDER-BOTTOM-COLOR: #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; = BORDER-RIGHT-COLOR: #d0d0d0 } .x-toolbar BUTTON { COLOR: #444 } .x-toolbar .x-btn-menu-arrow-wrap .x-btn-center BUTTON { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/btn-arrow.gif) } .x-toolbar .x-btn-text-icon .x-btn-menu-arrow-wrap .x-btn-center BUTTON = { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/btn-arrow.gif) } .x-toolbar .x-btn-over .x-btn-left { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-over .x-btn-right { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-over .x-btn-center { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-over BUTTON { COLOR: #111 } .x-toolbar .x-btn-click .x-btn-left { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-pressed .x-btn-left { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-menu-active .x-btn-left { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-click .x-btn-right { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-pressed .x-btn-right { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-menu-active .x-btn-right { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-click .x-btn-center { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-pressed .x-btn-center { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .x-btn-menu-active .x-btn-center { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/toolbar/tb-btn-sprite.gif) } .x-toolbar .ytb-sep { BACKGROUND-IMAGE: = url(/core/extjs/ext-2.1/resources/images/default/grid/grid-split.gif) } .x-tab-panel-header { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND: #eaeaea; BORDER-BOTTOM-COLOR: = #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-panel-footer { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND: #eaeaea; BORDER-BOTTOM-COLOR: = #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-panel-header { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-panel-footer { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } UL.x-tab-strip-top { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND: = url(/core/ext.ncbi/1/img/oldentrez/tabs/tab-strip-bg.gif) #dbdbdb = repeat-x left top; BORDER-BOTTOM-COLOR: #d0d0d0; BORDER-TOP-COLOR: = #d0d0d0; PADDING-TOP: 2px; BORDER-RIGHT-COLOR: #d0d0d0 } UL.x-tab-strip-bottom { BORDER-LEFT-COLOR: #d0d0d0; BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/tabs/tab-strip-btm-bg.gif); = BORDER-BOTTOM-COLOR: #d0d0d0; BORDER-TOP-COLOR: #d0d0d0; = BORDER-RIGHT-COLOR: #d0d0d0 } .x-tab-strip SPAN.x-tab-strip-text { COLOR: #333 } .x-tab-strip-over SPAN.x-tab-strip-text { COLOR: #111 } .x-tab-strip-active SPAN.x-tab-strip-text { COLOR: #333 } .x-tab-strip-disabled .x-tabs-text { COLOR: #aaaaaa } .x-tab-strip-top .x-tab-right { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/tabs/tabs-sprite.gif) } .x-tab-strip-top .x-tab-left { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/tabs/tabs-sprite.gif) } .x-tab-strip-top .x-tab-strip-inner { 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elmnts.length; i < len; i++)=0A= {=0A= if (elmnts.item(i).className.indexOf(className, " ") = !=3D -1)=0A= {=0A= elmnts.item(i).innerHTML =3D innerHTML;=0A= unObscuredEmails[className] =3D true=0A= =0A= }=0A= }=0A= }=0A= }=0A= catch(e){=0A= }=0A= }=0A= =0A= //=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=0A= function pubMedDbLinkSubmit(control) {=0A= try{=0A= if (control.pubmedOption)=0A= control =3D control.pubmedOption=0A= if (control.options)=0A= location.href =3D = control.options[control.options.selectedIndex].value;=0A= }=0A= catch(e){}=0A= return false;=0A= }=0A= =0A= //=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= 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href if 'redirect-to-url' is = missing in ref.=0A= if (typeof (ref) !=3D 'undefined' && = ref.indexOf('redirect-to-url') =3D=3D -1)=0A= {=0A= ref =3D ref + '&redirect-to-url=3D' + = encodeURIComponent(href)=0A= //=0A= jQuery.ajax({=0A= type: "HEAD",=0A= url: report_url + '?' + ref,=0A= timeout: 1000,=0A= async: false=0A= });=0A= }=0A= =0A= return true;=0A= });=0A= };=0A= =0A= // function to replace unsupported charachters in author names to be = searchable in PubMed database.=0A= function replacePubMedUnsupportedChars (str)=0A= {=0A= // replace low 128 chars automatically=0A= str =3D str.replace(/%26%23(x000[0-7][0-9a-f])%3b/ig,=0A= function($0, $1){=0A= return (String.fromCharCode('0' + $1))=0A= }=0A= )=0A= // replace others based on map and if it is not in the map replace = with empty string.=0A= str =3D = str.replace(/%26%23(x[0-9a-f][0-9a-f][0-9a-f][0-9a-f][0-9a-f])%3b/ig,=0A= function($0, $1){=0A= return (pubmedUnsupportedCharMap[$1] ? = pubmedUnsupportedCharMap[$1] : '')=0A= }=0A= )=0A= return str;=0A= }=0A= //=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=0A= // Based on script written by Dean Edwards, 2005=0A= // http://dean.edwards.name/=0A= =0A= var pmc_script =3D {=0A= addEvent: function(element, type, handler) {=0A= // assign each event handler a unique ID=0A= if (!handler.$$guid) handler.$$guid =3D this.addEvent_guid++;=0A= // create a hash table of event types for the element=0A= if (!element.events) element.events =3D {};=0A= // create a hash table of event handlers for each element/event pair=0A= var handlers =3D element.events[type];=0A= if (!handlers) {=0A= handlers =3D element.events[type] =3D {};=0A= // store the existing event handler (if there is one)=0A= if (element["on" + type]) {=0A= handlers[0] =3D element["on" + type];=0A= }=0A= }=0A= // store the event handler in the hash table=0A= handlers[handler.$$guid] =3D handler;=0A= // assign a global event handler to do all the work=0A= element["on" + type] =3D handleEvent;=0A= =0A= =0A= function handleEvent(event) {=0A= var returnValue =3D true;=0A= // grab the event object (IE uses a global event object)=0A= event =3D event || fixEvent(window.event);=0A= // get a reference to the hash table of event handlers=0A= var handlers =3D this.events[event.type];=0A= // execute each event handler=0A= for (var i in handlers) {=0A= this.$$handleEvent =3D handlers[i];=0A= if (this.$$handleEvent(event) =3D=3D=3D false) {=0A= returnValue =3D false;=0A= }=0A= }=0A= return returnValue;=0A= };=0A= =0A= function fixEvent(event) {=0A= // add W3C standard event methods=0A= event.preventDefault =3D fixEvent.preventDefault;=0A= event.stopPropagation =3D fixEvent.stopPropagation;=0A= return event;=0A= };=0A= =0A= fixEvent.preventDefault =3D function() {=0A= this.returnValue =3D false;=0A= };=0A= 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=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=3D=3D=0A= var hoverHandlers =3D function (opts) {=0A= this.config =3D {}=0A= this.self =3D this;=0A= this.__init(opts)=0A= }=0A= =0A= $j.fn.extend(hoverHandlers.prototype, {=0A= //=0A= __init: function(opts) {=0A= $j.fn.extend(this.config, opts)=0A= },=0A= //=0A= over: function (el) {=0A= var canvas =3D $j('.large-thumb-canvas', el)=0A= var prnt =3D canvas.parent();=0A= var win =3D $j(window)=0A= =0A= //=0A= if (canvas && prnt && win)=0A= {=0A= =0A= // collect necessary coordinates from all = intresting objects=0A= canvas.coords =3D this._x_coords(canvas, = 'canvas')=0A= prnt.coords =3D this._x_coords(prnt, = 'parent')=0A= win.coords =3D this._x_coords(win, = 'window')=0A= =0A= // replace @src with value of @hires, because = href hold high resolution image and src low one.=0A= $j('img', canvas).attr('src',=0A= function(){=0A= var hires =3D $j(this).attr("hires");=0A= return (typeof (hires) !=3D = 'undefined' && hires !=3D this.src)=0A= ? hires : this.src=0A= }=0A= )=0A= =0A= // make arrangement of canvas in the view port = nearby parent element.=0A= var cnvsRltvToPrnt =3D = this._x_arrange_canvas(canvas.coords, prnt.coords, win.coords)=0A= =0A= // visualize the canvas with images.=0A= canvas.css('top', cnvsRltvToPrnt.top + 'px')=0A= canvas.css('left', cnvsRltvToPrnt.left + 'px')=0A= canvas.css('display', 'block')=0A= } // canvas=0A= }, // over=0A= //=0A= out: function (el) {=0A= var canvas =3D $j('.large-thumb-canvas', el)=0A= canvas.css('display', 'none')=0A= },=0A= //=0A= _x_offset: function(el, elname) {=0A= var c =3D {}=0A= if (el[0] =3D=3D window )=0A= c =3D this._x_scroll(el, elname)=0A= else=0A= c =3D el.offset()=0A= //console.info('%s.offset {top,left} =3D {%s, %s}', = elname, c.top, c.left)=0A= return c=0A= },=0A= _x_scroll: function(el, elname) {=0A= var c =3D {top: el.scrollTop(), left: = el.scrollLeft()}=0A= //console.info('%s.scroll {top,left} =3D {%s, %s}', = elname, c.top, c.left)=0A= return c=0A= },=0A= _x_width: function(el, elname) {=0A= var w =3D el.outerWidth() || el.width()=0A= //console.info('%s.width =3D %s; outerWidth =3D = %s; width =3D %s', elname, w, el.outerWidth(), el.width())=0A= //IE: alert(elname + '.width =3D ' + w + '; = outerWidth =3D ' + el.outerWidth() + 'width =3D ' + el.width())=0A= return w=0A= },=0A= _x_height: function(el, elname) {=0A= var h =3D el.outerHeight() || el.height()=0A= // console.info('%s.height =3D %s', elname, h)=0A= return h=0A= },=0A= _x_coords: function(el, elname) {=0A= var size =3D { width: this._x_width (el, elname), = height: this._x_height(el, elname)}=0A= var coords =3D {=0A= scroll: this._x_scroll(el, elname),=0A= width: size.width,=0A= height: size.height,=0A= offset: this._x_offset(el, elname),=0A= size: size=0A= }=0A= return coords=0A= },=0A= _x_get_rltv_coord: function(trgtObj, baseObj, = label) {=0A= var c =3D {top: trgtObj.top - baseObj.top, left: = trgtObj.left - baseObj.left}=0A= //console.info("%s {top: %s, left: %s}", label, = c.top, c.left)=0A= return c=0A= },=0A= //=0A= _x_get_empty_space: function(objCoords, = objSize, viewPortCoords, viewPortSize, label) {=0A= var objRelToViewPort =3D = this._x_get_rltv_coord(objCoords, viewPortCoords, '_x_get_empty_space: = for ' + label);=0A= var c =3D {above: objRelToViewPort.top,=0A= below: viewPortSize.height - = (objRelToViewPort.top + objSize.height),=0A= left: objRelToViewPort.left,=0A= right: viewPortSize.width - = (objRelToViewPort.left + objSize.width)}=0A= $j.each(c, function(k, v){c[k] =3D (v < 0 ? 0 : v)})=0A= //console.info("%s {above: %s, below: %s, left: %s, = right: %s}", label, c.above, c.below, c.left, c.right)=0A= return c=0A= },=0A= //=0A= _x_get_bigest_space_area_name: function(spaces, label) {=0A= var sArr =3D [];=0A= for (k in spaces){ sArr[sArr.length] =3D {k: k, v: = spaces[k]} }=0A= // sort in reverse order=0A= sArr.sort (function(a,b){return ( a.v < b.v ? 1 : ( = a.v > b.v ? - 1 : 0 ) )})=0A= // console.info('%s: %s', label, sArr[0].k)=0A= return sArr[0].k=0A= },=0A= //=0A= _x_get_space_ratios: function(spaces, = viewPortSize, label) {=0A= var ratios =3D {above: 0, left: 0}=0A= if (viewPortSize.height > 0)=0A= ratios.above =3D spaces.above / = viewPortSize.height=0A= if (viewPortSize.width > 0)=0A= ratios.left =3D spaces.left / viewPortSize.width=0A= // console.info('ratio: %s {above: %s, left: %s}', = label, ratios.above, ratios.left)=0A= return ratios=0A= },=0A= _x_arrange_canvas: function(c_c, prnt_c, = viewp_c) {=0A= // then calculate empty space areas around parent = element of the canvas, which is ususally an anchor ("a" element)=0A= var emptySpacesArndPrntInViewPort =3D = this._x_get_empty_space (prnt_c.offset, prnt_c.size, viewp_c.offset, = viewp_c.size, 'emptySpacesArndPrntInWinPort')=0A= =0A= // get the area name, where the most linear space = avaiable=0A= var areaName =3D = this._x_get_bigest_space_area_name(emptySpacesArndPrntInViewPort, = viewp_c.size, 'spacios areaName')=0A= =0A= // calculate the proportions of area to be taken by = canvas in empty space area=0A= // relativly to the parent element.=0A= var ratios =3D = this._x_get_space_ratios(emptySpacesArndPrntInViewPort, viewp_c.size, = 'ratios')=0A= =0A= // calculate parent's coordinates relative to view = port=0A= var prntRltvToViewPort =3D = this._x_get_rltv_coord(prnt_c.offset, viewp_c.offset, = 'prntRltvToViewPort')=0A= =0A= // declare canvas relative coordinates=0A= var cnvsRltvToPrnt =3D {top: 0, left: 0}=0A= =0A= // choose appropriate location for placing canvas.=0A= // and choose the 1st element of an array as the one = with the biggest area around parent of canvas.=0A= // console.info ('place it at the %s', = bigestEmptySpaceAreaName)=0A= // calculations are relative to the left corner of = the parent element of canvas. (it has to have the css property = position:relative)=0A= switch (areaName)=0A= {=0A= case 'above':=0A= cnvsRltvToPrnt.top =3D - c_c.height=0A= cnvsRltvToPrnt.left =3D prnt_c.width * 0.5 - = c_c.width * ratios.left=0A= break;=0A= case 'below':=0A= cnvsRltvToPrnt.top =3D prnt_c.height=0A= cnvsRltvToPrnt.left =3D prnt_c.width * 0.5 - = c_c.width * ratios.left=0A= break;=0A= case 'left':=0A= cnvsRltvToPrnt.left =3D - c_c.width=0A= cnvsRltvToPrnt.top =3D prnt_c.height * 0.5 = - c_c.height * ratios.above=0A= break;=0A= case 'right':=0A= cnvsRltvToPrnt.left =3D prnt_c.width=0A= cnvsRltvToPrnt.top =3D prnt_c.height * 0.5 = - c_c.height * ratios.above=0A= break;=0A= }=0A= =0A= // recalculate canvas' coordinates relative to = parent into coordinates relative to view port.=0A= var cnvsRltvToViewPort =3D {=0A= top: prntRltvToViewPort.top + = cnvsRltvToPrnt.top,=0A= left: prntRltvToViewPort.left + = cnvsRltvToPrnt.left=0A= }=0A= //console.info("cnvsRltvToPrnt {top:%s, left: %s}", = cnvsRltvToPrnt.top, cnvsRltvToPrnt.left)=0A= //console.info("cnvsRltvToViewPort {top:%s, left: = %s}", cnvsRltvToViewPort.top, cnvsRltvToViewPort.left)=0A= =0A= // adjust relative position of canvas to the window = if calculated values=0A= // are forcing canvas go out of view port=0A= // next order should be preserved to keep left top = corner visible always=0A= =0A= // adjust right side and move a bit to the left=0A= if (cnvsRltvToViewPort.left + c_c.width > = viewp_c.width)=0A= {=0A= var horAdj =3D c_c.width - (viewp_c.width - = cnvsRltvToViewPort.left)=0A= cnvsRltvToPrnt.left -=3D horAdj=0A= cnvsRltvToViewPort.left -=3D horAdj=0A= //console.log('adjust right side and move to = the left %spx', horizontalAdjustment)=0A= }=0A= =0A= // adjust below side and move to the top=0A= if (cnvsRltvToViewPort.top + c_c.height > = viewp_c.height)=0A= {=0A= var vertAdj =3D c_c.height - (viewp_c.height - = cnvsRltvToViewPort.top)=0A= cnvsRltvToPrnt.top -=3D vertAdj=0A= cnvsRltvToViewPort.top -=3D vertAdj=0A= // console.log('adjust below side and move to = the top %spx', verticalAdjustment)=0A= }=0A= =0A= // adjust left side and move to the right=0A= if (cnvsRltvToViewPort.left < 0)=0A= {=0A= // console.log('adjust left side and move to the = right %spx', -(cnvsLeftRelativeToWindowPort))=0A= cnvsRltvToPrnt.left -=3D = cnvsRltvToViewPort.left=0A= cnvsRltvToViewPort.left =3D 0=0A= }=0A= // adjust above side and move to the bottom=0A= if (cnvsRltvToViewPort.top < 0)=0A= {=0A= // console.log('adjust above side and move to = the bottom %spx', -(cnvsTopRelativeToWindowPort))=0A= cnvsRltvToPrnt.top -=3D = cnvsRltvToViewPort.top=0A= cnvsRltvToViewPort.top =3D 0=0A= }=0A= // console.info("cnvsRltvToPrnt adjusted {top: %s, = left: %s}", cnvsRltvToPrnt.top, cnvsRltvToPrnt.left)=0A= // console.info("cnvsRltvToViewPort adjusted {top: = %s, left: %s}", cnvsRltvToViewPort.top, cnvsRltvToViewPort.left)=0A= return cnvsRltvToPrnt=0A= }=0A= })=0A= // = =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D end of hoverHandlers = =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=0A= =0A= =0A= // = =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D $j.fn.figPopup = =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=0A= function hoverOverHandler () {=0A= var h =3D new hoverHandlers();=0A= h.over(this)=0A= }=0A= =0A= function hoverOutHandler () {=0A= var h =3D new hoverHandlers();=0A= h.out(this)=0A= }=0A= =0A= $j.fn.figPopup =3D function(opts){=0A= var config =3D {=0A= interval: 500,=0A= timeout: 0,=0A= over: hoverOverHandler, // handler onMouseOver=0A= out: hoverOutHandler // handler onMouseOut=0A= }=0A= $j.extend(config, opts)=0A= return config=0A= }=0A= =0A= // = =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D end of $j.fn.figPopup = =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=0A= =0A= }) (jQuery) // end of extention of jQuery with figPopup=0A= =0A= =0A= =0A= jQuery(document).ready( function () {=0A= var fp_cfg =3D new jQuery.fn.figPopup()=0A= jQuery('a.figpopup').hoverIntent(fp_cfg)=0A= });=0A= }=0A= =0A= // end of hoverIntent routines for figures' popups=0A= // = *************************************************************************= ***=0A= // implementation of ....=0A= // = *************************************************************************= ***=0A= ------=_NextPart_000_0000_01CAD4DC.9F0D8F00 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://www.ncbi.nlm.nih.gov/corehtml/pmc/js/noext.menu.js if (typeof(noext) =3D=3D=3D 'undefined') { noext =3D {};=20 } if (typeof jQuery =3D=3D=3D 'undefined') { throw new Error('noext.Menu: jQuery must be present'); } // Constructor noext.Menu =3D function (n, subMenuStyle) { this.n =3D n; this.subMenuStyle =3D (subMenuStyle =3D=3D=3D 'ext') ? 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sm.addClass(subMenuStyle); } else { sm.removeClass(subMenuStyle); } e.stopPropagation(); // don't let default event occur = (removeMenus from document.click) =20 }, hover: function () { jQuery(this).addClass('active-item'); }, unHover: function () { jQuery(this).removeClass('active-item'); }, closeOtherSubMenus: function (currentSubMenu) { // close all menu's submenus, that are different than currently = clicked menu's submenu var subMenuStyle =3D this.subMenuStyle; currentSubMenu =3D this.subMenu; var menuInstances =3D noext.Menu.menuInstances; for (var i =3D 0; i < menuInstances.length; i++) { var menuInstance =3D menuInstances[i]; =20 if (currentSubMenu !=3D=3D menuInstance.subMenu && = menuInstance.subMenu.hasClass(subMenuStyle)) { menuInstance.subMenu.removeClass(subMenuStyle); }=20 } }, closeAllSubMenus: function (event) { // for when user clicks on document var menuInstances =3D noext.Menu.menuInstances; for (var i =3D 0; i < menuInstances.length; i++) { var subMenu =3D 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