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Transl Oncol. 2009 December 1; = 2(4):=20 247=E2=80=93257.
PMCID:=20 = PMC2781066
Copyright=20 =C2=A9 2009 Neoplasia Press, Inc. All rights reserved
Brain Tumor Stem-Like Cells Identified by = Neural Stem=20 Cell Marker CD15
Xing-gang Mao,* = Xiang=20 Zhang,* Xiao-yan Xue,=E2=80=A0 Geng = Guo,* Peng=20 Wang,* Wei Zhang,* Zhou Fei,* = Hai-ning=20 Zhen,* Si-wei You,=E2=80=A1 and Hao = Yang=E2=80=A1
*Department of Neurosurgery, = Xijing=20 Hospital, The Fourth Military Medical University, Xi'an, Shaanxi = Province,=20 China
=E2=80=A0Department of = Pharmacology, The Fourth=20 Military Medical University, Xi'an, Shaanxi Province, China
=E2=80=A1Institute of = Neurosciences, The Fourth=20 Military Medical University, Xi'an, Shaanxi Province, China
Address all = correspondence to:=20 Xiang Zhang, Department of Neurosurgery, Xijing Hospital, The = Fourth=20 Military Medical University, Xi'an, Shaanxi Province, China, = 710032.=20 E-mail: xzhang/at/fmmu.edu.cn
Received February 27, 2009; Revised April = 22, 2009;=20 Accepted April 29, 2009.
Abstract
In recent years, a = small number=20 of cells that have stem cell properties were identified in human = gliomas=20 called brain tumor stem cells (BTSCs), which were thought to = mainly=20 contribute to the initiation and development of gliomas and could = be=20 identified by the surface marker CD133. However, recent studies = indicated=20 that the expression of CD133 might be regulated by environmental=20 conditions such as hypoxia and that there might be = CD133-=20 BTSCs. Genetic mouse models demonstrated that some gliomas = originated from=20 transformed neural stem cells (NSCs). Therefore, we investigated = the=20 expression of CD15, a surface marker for NSCs, in tumor spheres = derived=20 from astrocytoma and ependymoma. CD15+ cells isolated = from=20 these tumor spheres had properties of BTSCs including = self-renewal,=20 multidifferentiation, and the ability to recapitulate the = phenocopy of=20 primary tumors. CD15 exhibited stable expression in long-term = cultured=20 tumor spheres, which sustained BTSCs properties, whereas CD133 = expression=20 decreased significantly in late passages. Furthermore,=20 CD15+CD133- cells isolated from early or = late=20 passages of tumor spheres showed similar characteristics of BTSCs. = Examination of glioma samples by immunohistochemistry showed that = CD15 was=20 expressed in a subset of human brain tumors. Therefore, CD15 can = be used=20 as a marker of stem-like cells derived from brain tumors that = might=20 contain CD133- BTSCs.
Introduction
The identification of = brain tumor=20 stem cells (BTSCs) marks a step toward finding new and effective = ways to=20 treat malignant brain tumors, one of the most lethal cancers = afflicting=20 both children and adults [1]. The concept of BTSCs has constructive = significance for=20 clinical practices because it has been elucidated that BTSCs = contribute to=20 relapse and chemoresistance or radioresistance of brain tumors [2=E2=80=934]. To date,many studies exploring = the property of=20 BTSCs built on the assumption that BTSCs express a cell surface = marker,=20 CD133 [5,6]. However, it has been indicated that = expression of=20 CD133 could be regulated by environmental conditions such as = hypoxia [7], and contrary results have been reported that = there are=20 CD133- BTSCs [8,9]. The existence of both CD133-positive and = -negative=20 BTSCs implies that further characterization of BTSCs is of = tremendous=20 interest. This also implies that one persistent challenge is our = inability=20 to recognize BTSCs, and many issues about the BTSCs are to be = answered.=20 For example, what is the significance of CD133 expression in = BTSCs? Are=20 there other markers that can specifically identify BTSCs? What is = the=20 relationship between BTSCs and neural stem cells (NSCs)? Is glioma = derived=20 from ancestor BTSCs or are BTSCs emerged after the forming of=20 tumors?
The identification of NSCs = provided new=20 possible targets of tumorigenic transformation of gliomas [10]. In fact, many evidences support the idea that = gliomas=20 are derived from transformed NSCs [11]. Recent studies using genetic mouse models = suggested=20 that at least a portion of gliomas were originated from = transformed NSCs,=20 which have the characteristics of reported BTSCs and are = responsible for=20 the formation of tumors [12,13]. In addition, many functional and = molecular=20 similarities have been elucidated between BTSCs and normal NSCs. = Both=20 BTSCs and NSCs have immortal proliferative potential in the = presence of=20 epidermal growth factor (EGF) and basic fibroblast growth factor = (bFGF)=20 and can differentiate into neuronal and glia lineages when = withdrawing=20 these growth factors and adding serum [6,14,15]. Both of them have an = environmental niche=20 that is important for their maintenance of stemness [16,17]. Many signaling pathways critical for = NSCs are=20 also important for BTSCs, but some of them were aberrantly = regulated [4,18]. These facts imply that it would be = necessary to=20 study the relationship between NSCs and BTSCs, and importantly, = BTSCs=20 might retain some properties of NSCs.
NSCs in adult brains can = be=20 recognized by the surface marker CD15 [19]. CD15 (leukocyte cluster of differentiation = 15), which=20 is the trisaccharide 3-fucosyl-N-acetyllactosamine or = FAL, also=20 known as stage-specific embryonic antigen 1 or leX antigen, is = highly=20 expressed in many types of pluripotent stem cells [19]. Recent studies suggested that, besides the=20 traditionally recognized NSCs that reside in the subventricular = zone,=20 CD133+/CD24- cells, but not CD24+ = cells=20 in the ependyma represent another population of NSCs [20]. Therefore, it is more likely that there are = at least=20 two types of NSCs in normal brains identified by CD133 and CD15. = However,=20 to our knowledge, although CD133 has been widely studied, the = expression=20 of CD15 has not been fully explored in BTSCs. Previous studies = focused on=20 the expression of CD15 in gliomas got contradictory results [21=E2=80=9324], and studies on BTSCs = showed that=20 glioma-derived neural spheres expressed CD15 [15]. Given the similarity between NSCs and BTSCs = and recent=20 disputes about CD133 as a BTSCs marker, we performed this study to = further=20 explore the expression of CD15 in BTSCs and to investigate if CD15 = can be=20 used to identify a group of BTSCs different from CD133+ = cells.
Materials and Methods
Glioma Samples
Seven glioma = samples used for=20 cell culture were provided by the Department of Neurosurgery, = Xijing=20 Hospital, the Fourth Military Medical University, including one = grade 2=20 oligoastrocytoma, two anaplastic astrocytoma (World Health = Organization=20 [WHO] grade 3), three glioblastoma (WHO grade 4), and one = ependymoma (WHO=20 grade 2; Table 1). = Additional 54=20 samples were used to investigate CD15 expression in gliomas by=20 Immunohistochemical analysis. Tumors were histopathologically = classified=20 according to the WHO classification. Informed consent was obtained = from=20 each patient, and experiments were approved by the local ethics=20 committee.
Culture of Primary Glioma = Spheres
Samples were stored = in=20 Dulbecco's modified Eagle's medium (DMEM)-F12 culture medium = (Invitrogen,=20 Carlsbad, CA) and processed within 1 hour after resection. Tumor = samples=20 were washed by DMEM-F12 medium and incubated for 20 minutes at = 37=C2=B0C in=20 TrypLE (Invitrogen). Then the tumor samples were dissociated to a=20 single-cell suspension using a fire-polished Pasteur pipette. The=20 dissociated tumor cells were centrifuged for 5 minutes at = 800g=20 and resuspended in either serum-free medium (SFM) consisting of = DMEM-F12=20 medium, EGF (20 ng/ml; Invitrogen), bFGF (20 ng/ml; Invitrogen), = and B27=20 (1:50; Invitrogen) or serum-containing medium (SCM) consisting of = DMEM-F12=20 medium with 10% fetal bovine serum (Gibco BRL Life Technologies,=20 Rockville, MD).
Limiting Dilution Assay and = Primary=20 Sphere Formation Assay
Limiting dilution = assay was=20 performed as described previously [25]. Tumor spheres were washed and dissociated to = a=20 single-cell suspension as described above. Then dissociated tumor = cells=20 were resuspended in DMEM-F12 medium to assess viable cell numbers = by=20 Trypan Blue (Sigma, St Louis, MO) exclusion. Acutely dissociated = tumor=20 cells were plated in 96-well microwell plates in 0.2 ml of SFM. = Final cell=20 dilutions ranged from 200 cells per well to 1 cell per well. = Cultures were=20 fed 0.025 ml of SFM every 2 days until day 14. The percentage of = wells not=20 containing spheres for each cell plating density were calculated = and=20 plotted against the number of cells per well. The number of cells = required=20 to form one tumor sphere, which reflected the proportion of tumor = stem=20 cells in the cell population, was then determined from the point = at which=20 the line crossed the 0.37 level. For primary sphere formation = assays, this=20 analysis was performed on the entire acutely dissociated tumor = cell=20 population to quantify stem cell frequency within the = tumor.
Cell Proliferation Assays
Tumor spheres were = dissociated=20 and plated in 96-well microwell plates in 0.2-ml volumes of SFM=20 supplemented with growth factors at a density of 2000 cells per = well.=20 Tumor spheres were collected from six wells every 2 days after = plating and=20 dissociated to get single cells as mentioned previously to assess = the=20 total number of cells.
Differentiation Assay of Tumor=20 Spheres
Tumor spheres were = plated onto=20 glass coverslips coated with poly-l-lysine=20 (Sigma) in SCM in individual wells of a 24-well culture plate. = Cells were=20 fed with SCM every 2 days, and coverslips were processed at = different days=20 after plating.
Immunofluorescence
Immunofluorescence = was=20 performed on primary brain tumor stem-like cells, serum-cultured = cells,=20 and glioma cell lines as described previously [5]. Briefly, for immunostaining of undifferentiated = tumor=20 spheres, cells were plated onto poly-l-lysine-coated glass coverslips in SCM = for 4=20 hours. Cells were then fixed with 4% paraformaldehyde and = incubated with=20 the following antibodies: CD133/1 (1:50, mouse monoclonal = immunoglobulin=20 G1 [IgG1]; Miltenyi Biotec, Bergisch Gladbach, Germany), CD15 = (1:150, MMA,=20 mouse monoclonal; BD Biosciences, San Jose, CA), Nestin (1:100, = 10C2,=20 mouse monoclonal; Abcam, Cambridge, MA), =CE=B2-III-tubulin = (1:500, mouse=20 monoclonal IgG1; Abcam), NeuN (1:1000, mouse monoclonal IgG1; = Chemicon,=20 Temecula, CA), glial fibrillary acidic protein (GFAP, 1:5000, = rabbit=20 polyclonal; Abcam), Galc (1:200, rabbit polyclonal; Chemicon), and = Ki67=20 (1:100, rabbit monoclonal; Abcam). Appropriate secondary = antibodies (Texas=20 Red goat antirabbit and Alexa 488 goat antimouse; Molecular = Probes,=20 Invitrogen) were used. For immunostaining of differentiated tumor = cells,=20 immunofluorescence was performed on differentiated tumor spheres = cultured=20 in SCM. For immunostaining of serum-cultured cells and glioma cell = lines,=20 serum-cultured cells and U87, U251, and SHG44 glioma cells were = plated=20 onto glass coverslips for 24 hours, and immunocytochemistry was = performed=20 as described previously. Cells were additionally counterstained = with=20 Hoechst 33342 (Sigma) to permit counting of cell nuclei. = Quantification of=20 cells stained with each antibody was averaged and estimated as a=20 percentage of total nuclei counted.
Isolation of CD15+ = and=20 CD15+CD133- Cells
CD15+ = and=20 CD15+CD133- cells were isolated by magnetic = bead=20 sorting to investigate their capacity to form tumor spheres. = Cultured=20 tumor spheres were dissociated to get single-cell suspensions as = described=20 previously. Then, the dissociated cells were centrifuged at = 800g=20 for 5 minutes. Cells were incubated with anti-CD15 antibody and=20 biotin-conjugated secondary antibody. Then biotin-labeled cells = were=20 rinsed and labeled by streptavidin-coupled Dynabeads (Dynal; = Invitrogen)=20 for 30 minutes. Cells were rinsed with phosphate-buffered saline = during=20 each step. Magnetic separation was carried out on the Dynal magnet = (Dynal;=20 Invitrogen) to separate CD15+ and CD15- = cells. For=20 CD15+CD133- cells, CD133- cells = were=20 negatively selected. Then CD15+CD133- cells = were=20 isolated from these CD133- cells as described = previously. After=20 separation, tumor sphere formation and differentiation assays were = performed on CD15+, CD15-, and=20 CD15+CD133- cells.
Chromosome Analysis
Cells were treated = with 100 ml=20 of Colcemid (10 g/ml; Invitrogen) for 2 hours. Then cells were = resuspended=20 in hypotonic solution (75 mM KCl) for 30 minutes at 37=C2=B0C, = centrifuged, and=20 fixed in methanol and acetic acid. Metaphase spreads were prepared = on=20 slides, dried, and Giemsa-stained after trypsin = pretreatment.
Implantation into Nude = Mice
Single cells = dissociated from=20 primary tumor spheres, isolated CD15+, or = CD15-=20 cells were resuspended in 10 =C2=B5l of phosphate-buffered saline, = in aliquots=20 of 5000 or 10,000 cells. These aliquots were injected = stereotactically=20 into the right striatum of 6- to 8-week-old nude mice brains = (n =3D=20 6 each; Center of Experimental Animals, Fourth Military Medical=20 University, Xian, China), after administration of general = anesthesia. The=20 injection coordinates were 2 mm to the right of the midline, 0.5 = mm=20 anterior to the coronal suture, and 3 mm deep. The implanted mice = were=20 killed at 10 weeks after implantation by intracardiac = perfusion-fixation=20 with 4% paraformaldehyde and examined for tumors on the brain = sections.=20 All of the animals used were experimented in strict accordance = with the=20 animal experiments guidelines enforced at the Fourth Military = Medical=20 University.
Immunohistochemistry
For staining of = human and mouse=20 paraffin sections, tissue was fixed overnight in 4% = paraformaldehyde,=20 transferred to 70% ethanol, paraffin-embedded, and sectioned at a=20 thickness of 1 =C2=B5m. Sections were mounted on microscope slides = and then=20 dried overnight at 60=C2=B0C, dewaxed in xylene, and rehydrated = with distilled=20 water. Hematoxylin and eosin staining and immunohistochemical = staining of=20 the following antibodies were performed on the sections: = human-specific=20 Nestin (hNestin; 10C2, mouse monoclonal; Abcam), CD15, GFAP, and=20 =CE=B2-III-tubulin. With the exception of sections stained for = GFAP, all=20 sections were treated with heat-induced epitope retrieval = technique using=20 a citrate buffer at pH 6.0. Then the sections were blocked for = endogenous=20 peroxidase and biotin before the application of the primary = antibody.=20 Incubation of primary antibodies was performed for 3 hours at room = temperature. The Elite Vector Stain ABC System (Vector = Laboratories,=20 Burlingame, CA) was used as detection system and diaminobenzidine = was used=20 as a chromogen. The counterstain of preference was hematoxylin for = nuclear=20 detail.
Statistical Analysis
Statistical = analyses were=20 performed using Student's 2-tailed t test. P = < .05=20 was considered statistically significant. Statistical analyses = were done=20 using SPSS v.13.0.0 (SPSS, Inc, Chicago, = IL).
Results
Formation of Tumor = Spheres
SFM containing human = recombinant EGF=20 and bFGF were used to favor the growth of glioma spheres. Glioma = spheres=20 from the seven patients were presented as BTS1, BTS2, BTS3, BTS4, = BTS5,=20 BTS6, and BTS7, and the cells cultured in SCM were marked = correspondingly=20 as BT1, BT2, BT3, BT4, BT5, BT6, and BT7. Of the seven brain tumor = samples, numbers 1, 2, 4, 5, and 7 formed spheres within 1 month = in SFM=20 (Table 1). BTS1 = sustained=20 five passages and ceased growth. BTS2, BTS4, BTS5, and BTS7 have = been=20 cultured more than 10 passages (BTS2 and BTS5 for more than 20 = passages)=20 and were sustained now. However, tumor cells from samples 3 and 6 = did not=20 form tumor spheres within 1 month. Of the formed tumor spheres, = BTS1,=20 BTS2, BTS5, and BTS7 grew as floating spheres, whereas BTS4 grew = as=20 adherent tumor spheres (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 1A3D"Figure),=20 consistent with previous report that some tumor spheres grew as = adherent=20 spheres [8]. The morphologies of the seven SCM cultured = cells were=20 also different from each other (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 1A3D"Figure).=20 When SCM-cultured tumor cells of late passages (passage 10) were=20 transformed to SFM, no tumor spheres were investigated within 1=20 month.
3D"Table<= /DIV>
Table=20 1
Summary = of Patients=20 Information.
Primary sphere formation = assay showed=20 that the stem cell frequencies in the investigated samples = (samples 1, 2,=20 4, 5, and 7) were between 3.7% and 20.1% (12.48% =C2=B1 6.53%). = Tumor spheres=20 cultured in SFM grew at a relatively slow rate at the first two = passages,=20 whereas they got a constant and greater growth rate after two = passages (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 1B3D"Figure).=20 Most SCM-cultured tumor cells showed a highly heterogeneous = morphology and=20 proliferated at constant rates. BT2 showed initial growth followed = by a=20 plateau phase, and then some cells got a much greater growth rate = and=20 proliferated dominantly at later passages (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 1C3D"Figure).=20 For ependymoma-derived tumor spheres that showed adherent growth = pattern,=20 BTS4 exhibited constant and a relatively slow growth rate (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 13D"Figure,=20 D and E).
Primary Tumor Spheres Cultured = in SFM=20 Showed BTSCs Properties
Besides BTS1, tumor = spheres=20 (BTS2, BTS4, BTS5, and BTS7) cultured in SFM could be serially = passaged=20 more than 10 times (Table 1), = indicating that=20 these stem-like cells have the potential of self-renewal, one = critical=20 feature of BTSCs. Immunofluorescence staining showed that both=20 astrocytoma- and ependymoma-derived spheres expressed stem cell = markers=20 CD133, Nestin, CD15 (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 23D"Figure,=20 A=E2=80=93F), and proliferative marker Ki67 (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 23D"Figure,=20 G and H). One property of BTSCs was their = potential to=20 differentiate in serum medium without growth factors. Therefore,=20 differentiation potential was examined in all cultured tumor = spheres.=20 Tumor spheres of BTS1, BTS2, BTS4, BTS5, and BTS7 were plated onto = poly-l-lysine-coated coverslips = and cultured=20 in SCM. After culturing in this differentiation medium, all tumor = spheres=20 adhered and cells migrated from the spheres (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 3A3D"Figure).=20 Immunofluorescence staining showed that neuronal and glial lineage = markers=20 NeuN, =CE=B2-III-tubulin, GFAP, and Galc were expressed in the = differentiated=20 cells. However, a considerable portion of differentiated cells = expressed=20 both neuronal and glial markers, implying the aberrant = differentiation of=20 tumor stem-like cells (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 3B3D"Figure).
3D"Figure
<= STRONG>Figure=20 1
Morphologies and=20 proliferation kinetics of SFM- and SCM-cultured cells. (A)=20 Morphologies of SFM-cultured stem-like tumor spheres (BTS2 = and BTS4)=20 and serum-cultured cells (BT2 and BT4) from different = passages=20 (p2 indicates passage 2; p10, passage=20 10). = (more ...)
3D"Figure
<= STRONG>Figure=20 2
Expression of stem=20 cell markers and proliferate marker Ki67 in stem-like tumor = spheres.=20 (A=E2=80=93F) Tumor spheres BTS2 and BTS4 were shown as = representative to be=20 labeled with antibodies to CD133, Nestin, and CD15 (green). = Nuclei=20 were counterstained = (more ...)
CD15+ Cells Isolated = from=20 Tumor Spheres Showed Potential of BTSCs
It has been suggested = that CD15 was=20 a marker of NSCs. We detected the expression of CD15 in early = passages=20 (passage 2) of SFM-cultured tumor spheres. As a result, CD15 was = detected=20 in all the tumor spheres derived from the five samples that formed = stem=20 cell-like spheres (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 23D"Figure,=20 C and F), including BTS1 that sustained only = five=20 passages. Interestingly, although it was elucidated that CD15 was = not=20 expressed in normal ependyma cells [19], CD15 was detected in ependymoma-derived tumor = spheres=20 (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 2F3D"Figure).=20 Immunostaining for proliferative marker Ki67 showed that some=20 CD15+ cells were positive for Ki67 (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 23D"Figure,=20 G and H), indicating that brain tumor stem-like = cells=20 showed increased proliferative capacity [6].
Then we assessed if CD15 = can identify=20 stem cells in these tumor spheres. CD15-positive cells were = isolated by=20 magnetic bead sorting from BTS1, BTS2, BTS4, BTS5, and BTS7. Both=20 CD15-positive and CD15-negative cells were cultured in SFM to = investigate=20 their potential to form stem-like spheres. CD15+ cells = formed=20 tumor spheres within 1 week, whereas few CD15-negative cells = formed tumor=20 spheres within 2 weeks (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 33D"Figure,=20 C and D). The percentage of formed tumor spheres = in=20 CD15-positive and CD15-negative cells were 41.6% =C2=B1 3.2% and = 1.1% =C2=B1 0.4%,=20 respectively (P < .001). Tumor spheres formed by=20 CD15+ cells (CD15-TS) could be dissociated and form = secondary=20 tumor spheres, similar to their parental spheres. Besides CD15-TS = from=20 BTS1 that sustained three to four passages, CD15-TS from BTS2, = BTS4, BTS5,=20 and BTS7 were serially passaged more than five times, indicating = their=20 self-renewal capacity. When CD15-TS were induced to = differentiation, both=20 neuronal and glial cell markers =CE=B2-III-tubulin, NeuN, GFAP, = and Galc were=20 expressed (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 33D"Figure,=20 E=E2=80=93H). Like their parental tumor spheres, CD15-TS = showed aberrant=20 differentiation with many cells that expressed both neuronal and = glial=20 cell markers (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 33D"Figure,=20 I and J). Quantification of differentiated cells = showed=20 that percentages of GFAP, =CE=B2-III-tubulin, Galc-positive, and = GFAP,=20 =CE=B2-III-tubulin double-positive cells in differentiated cells = from CD15-TS=20 were similar to those of their parental tumor spheres (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 3B3D"Figure).
CD15+ Cells = Displayed=20 Tumor-Specific Properties
In addition to = forming=20 stem-like neural spheres in SFM, BTSCs were expected to bear some=20 tumor-specific features, such as unbalanced karyotypes. We = conducted=20 chromosome analysis on SFM- and SCM-cultured cells to demonstrate = that=20 cells in tumor spheres and CD15+ cells were indeed = transformed=20 and are not normal NSCs. For each SFM-cultured cell line (BTS2, = BTS4,=20 BTS5, and BTS7), identical abnormal numerical chromosomal = aberrations were=20 present in tumor sphere or CD15+ cells of both early = and late=20 passages (passages 2 and 10). As a representative, identical = numerical=20 chromosomal aberrations 47 XY, -10, +7, +21, were = detected in=20 BTS2 cells and CD15+ BTS2 cells of early and late = passages (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 4A3D"Figure).=20 However, for SCM-cultured tumor cells, more complicated numerical=20 chromosomal aberrations were present in late passages (data not = shown).=20 These results suggested that stem-like cells cultured in SFM = retained=20 their property with little changes, whereas tumor cells cultured = in SCM=20 showed chromosome instability at late passages [15].
3D"Figure
<= STRONG>Figure=20 3
Differentiation of=20 tumor spheres (BTS2 was shown as representative) in SCM. (A) = Cells=20 in tumor spheres cultured in SCM-migrated from the spheres = and=20 differentiated into glial and neuronal lineages. (B) = Differentiated=20 cells positive for each antigen and = (more ...)
CD15+ Cells Showed=20 Tumorigenesis In Vivo
CD15+ = cells were=20 isolated from expanded BTS2, BTS4, and BTS5. Then CD15+ = and=20 CD15- cells, SCM-cultured cells BT2, BT4, and BT5 were = injected=20 into nude mice brains, in aliquots of 5000 or 10,000 cells to = investigate=20 their potential to form tumors in vivo. The mice were = killed at=20 10 weeks after transplantation for examination of tumors. All of = the nude=20 mice that received CD15+ cells developed tumors (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 4B3D"Figure),=20 whereas none of the mice injected with SCM-cultured cells or=20 CD15- cells formed tumors. Anti-human Nestin-10C2 = antibody=20 (hNestin; Abcam), which does not react with mouse antigens, was = used to=20 specifically recognize human-derived cells expressing Nestin. = Brains of=20 normal nude mice were used as negative controls. hNestin was not = detected=20 in normal nude mice brain, indicating that Nestin-positive cells = detected=20 in xenografts would be derived from human tumor cells. = hNestin-positive=20 cells in the xenograft tumors demonstrated extensive infiltration = into the=20 surrounding cerebral cortex, a pathologic feature of human gliomas = (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 4C3D"Figure).=20 Furthermore, hNestin-positive cells showed a tendency for = migrating along=20 white matter tracts such as the corpus callosum (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 4D3D"Figure),=20 as is characteristically seen with glioma cells in the brains of = patients.=20 Immunohistologic staining showed that CD15 was expressed in both = primary=20 tumor samples and tumor xenografts (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 43D"Figure,=20 E and I). Examination of Nestin, GFAP, and = =CE=B2-III-tubulin=20 expression showed that primary and xenografts tumors had similar=20 immunohistologic characteristics (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 43D"Figure,=20 E=E2=80=93L). These data demonstrate that = CD15+ cells have the=20 potential to form tumors in vivo, a critical property of=20 BTSCs.
CD15 Expression Was Decreased = in=20 Differentiated Tumor Spheres
To further confirm if = CD15 was=20 predominantly expressed in stem-like tumor cells, we examined CD15 = expression in differentiated cells and serum-cultured cells (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 53D"Figure,=20 A=E2=80=93D). Decrease of CD15 expression was detected in = all=20 SFM-cultured cell lines after they were transformed to SCM. After = 4 days=20 of differentiation, CD15 was significantly decreased in the tumor = spheres=20 and was not expressed in the migrated cells (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5A3D"Figure).=20 After 14 days of differentiation, CD15 was almost not detected. = These=20 results meant that CD15 expression declined with the = differentiation of=20 BTSCs and was little or not expressed in more mature cells (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5E3D"Figure).
3D"Figure
<= STRONG>Figure=20 4
CD15+=20 cells showed aberrant karyotypes and tumorigenesis in = vivo=20 (data from BTS2 was shown as representative). (A) Chromosome = analysis demonstrated that CD15+ cells from BTS2 = showed=20 numerical chromosomal aberrations 47 XY, -10, +7, = +21.=20 Hematoxylin and = (more ...)
Primary tumor cells were = also=20 cultured in SCM. Although investigated in dissociated single cells = from=20 primary tumors (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5B3D"Figure),=20 CD15 was not detected in serum-cultured cells (BT1-BT7; <= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5C3D"Figure).=20 We also tested if standard glioma cell lines cultured in serum = medium=20 contain CD15+ cells. We performed immunostaining in = U87, U251,=20 and SHG44 cell lines, but CD15 was not detected in these cell = lines (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5D3D"Figure).=20 These results indicated that CD15 was not expressed in cells = cultured in=20 serum medium, in which stem-like cells were differentiated. These = results=20 also implied that serum-cultured cells and standard glioma cell = lines=20 shared many important properties different from SFM-cultured = stem-like=20 cells and primary tumors [15].
CD15 Identified BTSCs in Tumor = Spheres of=20 Late Passages
Because long-term = culture might=20 influence the expression of CD133, we also examined the expression = of=20 CD133 and CD15 in late passages of tumor spheres. In late passages = (passage 10), the percentage of CD133+ cells were = decreased=20 significantly, whereas decreased percentage of CD15+ = cells were=20 hardly appreciable (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 53D"Figure,=20 F and G). Limiting dilution assay showed that = stem-like=20 cell frequencies in early and late tumor spheres were similar (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5H3D"Figure).=20 In addition, differentiation assay showed that CD15-TS from early = and late=20 passages had similar potential to differentiate into neuronal and = glial=20 lineages (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 3B3D"Figure).=20 Together with the result that karyotype of BTSCs was stable in = late=20 passages, these facts suggested that SFM-cultured cells maintained = their=20 stem cell properties even in late passages and that decreased = percentage=20 of CD133+ cells in late passages might be, at least = partly, due=20 to the environmental factors as have been elucidated but not = necessarily=20 decreased stem cell potentials. Therefore, stable expression of = CD15 made=20 it more suitable to identify BTSCs from tumor spheres, especially = of late=20 passages.
CD15+ Cells Might = Contain=20 BTSCs That Are CD133 Negative
To further = investigate if=20 CD15+ cells represent one type of BTSCs different from=20 CD133+ cells, we isolated = CD15+CD133-=20 cells and tested if they have stem cell activities.=20 CD15+CD133- cells could be isolated from all = SFM-cultured tumor spheres of both early (passage 2) and late = passages=20 (passage 10, except BTS1). Tumor sphere formation assay and=20 differentiation assay showed that = CD15+CD133- cells=20 could also form tumor spheres in SFM, and cells in these tumor = spheres=20 differentiated into glial and neuronal lineages in SCM (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 53D"Figure,=20 I=E2=80=93K; <= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 3B3D"Figure).=20 Then CD15+ cells isolated from tumor spheres formed by=20 CD15+CD133- cells of late passages were = injected=20 into nude mice brains. Xenografts similar to those formed by early = CD15+ cells were investigated. Therefore, CD15 might = identify=20 BTSCs that contain CD133- cells from both early and = late=20 passages of tumor spheres.
CD15 Was Expressed in a Subset = of Human=20 Brain Tumors
To determine = whether CD15 is=20 expressed in human brain tumors, we performed immunohistochemical = analysis=20 on 54 archival brain tumor samples, including 20 grade 4 = glioblastomas, 11=20 grade 3 astrocytomas, 17 grade 2 astrocytomas, and 4 ependymomas.=20 Immunoreactivity for CD15 in granulocytes was detected in all = cases,=20 serving as an internal positive control (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 6A3D"Figure).=20 Primary antibody omission was used as negative control. CD15 was=20 investigated in a portion of all types of astrocytomas and = ependymomas (<= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure 63D"Figure,=20 B=E2=80=93F). This means a subset of brain tumors = contained=20 CD15+ stem- or progenitor-like cells, which might have=20 originated from transformed neural stem or progenitor cells and=20 responsible for the development of tumors.
3D"Figure
<= STRONG>Figure=20 5
CD15 = expression and=20 stem cell properties in differentiated and late-passage = tumor=20 spheres. (A) Expression of CD15 decreased significantly = after=20 differentiation. (B) CD15-positive cells were detected in = the=20 primary dissociated cells. Arrows in panels A = (more ...)
Discussion
In recent years, the = concept of=20 cancer stem cells (CSCs) gives us new insights about the tumor = biology.=20 The CSC hypothesis claimed that tumors were driven by a rear = population of=20 cells, CSCs, which have the potential of self-renewal,=20 multidifferentiation, and the ability to recapitulate the = phenotype of=20 origin tumors. Although there are controversies [26], the research about CSCs progressed rapidly in = recent=20 years and gave researchers new direction to find more effective = therapies.=20 For brain tumor, the identification of BTSCs leads to promising = progress=20 in treating gliomas [4,6,27]. However, some critical problems are = needed to=20 be solved to get substantial understanding of BTSCs. First, the = identity=20 of BTSCs is not clear. Present isolation methods, including = isolation=20 based on surface marker CD133 or side population, have led to = disputed=20 results of identifying BTSCs [8,28,29]. Second, the origin of BTSCs and = its=20 relationship with NSCs are not clear [30]. These problems mean that we only have little = knowledge=20 about BTSCs, and further studies are needed to explore the = characteristics=20 of BTSCs.
Previous studies showed that = single cells from=20 primary gliomas formed tumor spheres in SFM containing EGF and = bFGF. This=20 method has been used for many years to study NSCs. Only stem or = progenitor=20 cells survive in this medium, whereas differentiated mature cells = die, and=20 progenitor cells can only sustain limited passages. In recent = years, this=20 medium has been used to enrich stem cells from human brain tumors = [6,15]. In our study, tumor cells cultured in = SFM showed a=20 relatively slow growth rate within the first two passages and got = greater=20 and achieved a constant growth rate after two passages, possibly=20 reflecting the select process of this SFM culture system for = stem-like=20 cells. In the early passages, differentiated, more mature = progenitor and=20 other cells gradually disappeared, and the BTSCs pool survived and = proliferated. During late passages, most cells in tumor spheres = were stem=20 or progenitor cells and, therefore, showed constant growth rate. = As a=20 result, we got brain tumor spheres containing BTSCs from both = glioma and=20 ependymoma cultured in SFM. In our results, 5 (71.43%) in 7 = samples formed=20 tumor spheres within 1 month and 4 sustained more than 10 = passages.
Recent studies showed that not all = CD133-positive cells were BTSCs, and there were CD133-negative = BTSCs [8,9,28]. For example, A2B5+=20 CD133- brain tumor-initiating cells were identified = recently=20 [31]. In addition, the significance of CD133 = expression in=20 BTSCs was not clear [32]. It has long been hypothesized that brain = tumors might=20 have originated from NSCs [11]. Exploration of the mechanisms of glioma = tumorigenesis=20 using genetic mouse models implied that at least a part of the = gliomas=20 might have originated from transformed NSCs [12,13]. Therefore, we examined the expression = of CD15, a=20 marker of NSCs, in tumor spheres to assess if CD15 can be used to = enrich=20 BTSCs.
CD15 is expressed in many = pluripotent stem=20 cells, including pluripotent embryonic stem cells, in germinal = zones of=20 the developing CNS, on some astrocytes in the adult CNS, and = within adult=20 neurogenic zones. CD15 has been identified as one specific marker = for NSCs=20 [19] and, recently, as a marker for = tumor-propagating cells=20 in medulloblastoma [33]. Our results demonstrated that CD15 was = expressed in=20 both glioma- and ependymoma-derived tumor spheres and that its = expression=20 decreased dramatically in differentiated tumor spheres. In = addition, CD15=20 was not expressed in SCM-cultured cells and standard glioma cell = lines,=20 which have been suggested not containing stem cells. In fact,=20 serum-cultured cell lines were quite different from stem-like = cells in=20 many facets. For example, SFM-cultured stem-like cells, but not=20 serum-cultured primary cells or standard glioma cells lines (U251, = U87=20 cells, etc.), phenocopied the critically important histopathologic = features of the original human gliomas [15]. Another difference between SFM- and = SCM-cultured cells=20 was their stability of critical features. We analyzed the = chromosomes of=20 different passages of both SFM- and SCM-cultured cells and found = that=20 SFM-cultured tumor spheres largely maintained their initial = genotypes,=20 whereas, in contrast, SCM-cultured cells showed quite complex = changes of=20 genotypes. Together, these results indicated that CD15 was = expressed=20 mainly in stem-like brain tumor cells.
CD15 has been described as an NSC = marker that=20 did not contain the ependyma cells [19]. Recent studies elucidated that other types of = CD133-positive NSCs residing in the ependyma were different from=20 CD15+ NSCs because CD15 was not expressed in ependyma = cells [20,34]. These facts complicated the issue of = origin of=20 BTSCs and indicated that BTSCs may also contain different types of = =E2=80=9Cstem=20 cells,=E2=80=9D which probably originated from different types of = NSCs or=20 progenitors. It was not clear which type of NSCs, CD15+ = NSCs or=20 CD133+ NSCs, was more early, or if they represented = different=20 lineages of NSCs. Although we did not directly elucidate the = existence of=20 CD15+CD133+ cells, the percentage of=20 CD15+ cells and CD133+ cells in early = passages=20 (e.g., 76.5% =C2=B1 0.5% and 53.3% =C2=B1 4.1%, respectively, for = BTS2; <= SPAN=20 style=3D"TEXT-DECORATION: underline">Figure = 5E3D"Figure)=20 in tumor spheres indicated that there were=20 CD15+CD133+ BTSCs. This is different from = the=20 situations in normal nervous systems, where CD15 and CD133 were = expressed=20 by different cells. Whatever the origin of BTSCs and the = relationship=20 between CD15+ and CD133+ NSCs, both = expressions of=20 CD15 and CD133 in BTSCs implied that the identified stemlike cells = from=20 brain tumors did have the property of stem cells but were = aberrantly=20 transformed. In addition, the expression of CD15 in = ependymoma-derived=20 tumor spheres also implied the aberrant phenotype of these = stem-like tumor=20 cells.
Consistent with other reports [12,35], we investigated downregulation of = CD133 in late=20 passages of stem-like cells. In addition, CD133 might fail to = accurately=20 identify stem-like cells in late passages, whereas CD15 retained = its=20 property even at late passages. Recent studies discovering some = mechanisms=20 that regulate expression of CD133 in brain tumor cells indicated = that=20 expression of CD133 could be regulated by environmental conditions = such as=20 hypoxia and mitochondrial dysfunction [7]. The mechanisms of regulation of CD133 = expression in brain=20 tumors need to be further examined to elucidate their roles in = stem-like=20 cells.
The expression of CD15 in BTS1, = which=20 sustained only five passages, indicated that CD15 was also = expressed in=20 some progenitor cells. However, the existence of=20 CD15+CD133- BTSCs demonstrated that CD15 = might be=20 one marker for BTSCs, which contained CD133- cells. Our = results=20 and other reports indicated that CD133 was not expressed in all = brain=20 tumors (data not shown) [36,37]. Therefore, CD15 would be used as one = marker to=20 identify BTSCs from brain tumors that do not express CD133. The = expression=20 of the NSC marker CD15 in brain tumor stem-like cells implied some = intrinsic relationships between NSCs and BTSCs. The expression of = the NSCs=20 marker CD15 indicated that BTSCs might retain more characteristics = of NSCs=20 besides self-renewal and multidifferentiation.
These results raised = other questions=20 about NSCs and BTSCs such as the following: What is the = relationship=20 between CD15+ and CD133+ NSCs and BTSCs? How = have=20 CD15 and CD133 appeared during the formation of gliomas? Answers = to these=20 questions would lend credence to the identity of BTSCs and provide = novel=20 strategies to target these aberrantly transformed stem-like=20 cells.
Acknowledgments
The authors thank Juan Li and = Xiaoyan Chen for=20 their technical assistance in cell culture and = immunohistochemistry,=20 Zhenzhong Zhu for his generously provision of CD15 antibody, = Ganlan Bian=20 for her technical assistance in transplantation experiments, Hui = Xu for=20 her help in chromosome analysis, and Rong Cao, Jinmei Diao for = their=20 assistance in capturing images.
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Articles from Translational=20 Oncology are provided here courtesy of
Neoplasia Press
3D"Figure
<= STRONG>Figure=20 6
Expression of CD15=20 in human brain tumors. (A) CD15 was detected in = granulocytes,=20 serving as an internal positive control. (B=E2=80=93E) CD15 = was investigated=20 in glioblastoma (B), grade 3 astrocytoma (C), grade 2 = astrocytoma=20 (D), and ependymoma (E). (F) = (more ...)

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  • Mao,=20 X.
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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_010F_01CA8591.191A8140 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 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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: = url(../../img/oldentrez/tabs/scroll-right.gif); BORDER-BOTTOM-COLOR: = #aeaeae; BORDER-TOP-COLOR: #aeaeae; BORDER-RIGHT-COLOR: #aeaeae } .x-window-proxy { BORDER-LEFT-COLOR: #b0b0b0; BACKGROUND: #e0e0e0; BORDER-BOTTOM-COLOR: = #b0b0b0; BORDER-TOP-COLOR: #b0b0b0; BORDER-RIGHT-COLOR: #b0b0b0 } .x-window-tl .x-window-header { COLOR: #555 } .x-window-tc { BACKGROUND-IMAGE: url(../../img/oldentrez/window/top-bottom.png) } .x-window-tl { BACKGROUND-IMAGE: url(../../img/oldentrez/window/left-corners.png) } .x-window-tr { BACKGROUND-IMAGE: url(../../img/oldentrez/window/right-corners.png) } .x-window-bc { BACKGROUND-IMAGE: url(../../img/oldentrez/window/top-bottom.png) } .x-window-bl { BACKGROUND-IMAGE: url(../../img/oldentrez/window/left-corners.png) } .x-window-br { BACKGROUND-IMAGE: url(../../img/oldentrez/window/right-corners.png) } .x-window-mc { BORDER-RIGHT: #d0d0d0 1px solid; BORDER-TOP: #d0d0d0 1px solid; = BACKGROUND: #e8e8e8; BORDER-LEFT: #d0d0d0 1px solid; BORDER-BOTTOM: = #d0d0d0 1px solid } .x-window-ml { BACKGROUND-IMAGE: url(../../img/oldentrez/window/left-right.png) } .x-window-mr { BACKGROUND-IMAGE: url(../../img/oldentrez/window/left-right.png) } .x-panel-ghost .x-window-tl { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-collapsed .x-window-tl { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-window-plain .x-window-mc { BORDER-RIGHT: #eee 1px solid; BORDER-TOP: #d0d0d0 1px solid; = BACKGROUND: #e8e8e8; BORDER-LEFT: #d0d0d0 1px solid; BORDER-BOTTOM: #eee = 1px solid } .x-window-plain .x-window-body { BORDER-RIGHT: #d0d0d0 1px solid; BORDER-TOP: #eee 1px solid; = BACKGROUND: none transparent scroll repeat 0% 0%; BORDER-LEFT: #eee 1px = solid; BORDER-BOTTOM: #d0d0d0 1px solid } BODY.x-body-masked .x-window-mc { BACKGROUND-COLOR: #e4e4e4 } BODY.x-body-masked .x-window-plain .x-window-mc { BACKGROUND-COLOR: #e4e4e4 } .x-html-editor-wrap { BORDER-LEFT-COLOR: #d0d0d0; BORDER-BOTTOM-COLOR: #d0d0d0; = BORDER-TOP-COLOR: #d0d0d0; BORDER-RIGHT-COLOR: #d0d0d0 } .x-panel-noborder .x-panel-body-noborder { BORDER-TOP-WIDTH: 0px; BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: = 0px; BORDER-RIGHT-WIDTH: 0px } .x-panel-noborder .x-panel-header-noborder { BORDER-TOP-WIDTH: 0px; BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM: #d0d0d0 = 1px solid; BORDER-RIGHT-WIDTH: 0px } .x-panel-noborder .x-panel-tbar-noborder .x-toolbar { BORDER-TOP-WIDTH: 0px; BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM: #d0d0d0 = 1px solid; BORDER-RIGHT-WIDTH: 0px } .x-panel-noborder .x-panel-bbar-noborder .x-toolbar { BORDER-TOP: #d0d0d0 1px solid; BORDER-LEFT-WIDTH: 0px; = BORDER-BOTTOM-WIDTH: 0px; BORDER-RIGHT-WIDTH: 0px } .x-window-noborder .x-window-mc { BORDER-TOP-WIDTH: 0px; BORDER-LEFT-WIDTH: 0px; BORDER-BOTTOM-WIDTH: = 0px; BORDER-RIGHT-WIDTH: 0px } .x-window-plain .x-window-body-noborder { BORDER-TOP-WIDTH: 0px; BORDER-LEFT-WIDTH: 0px; 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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 { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/tabs/tabs-sprite.gif) } .x-tab-strip-bottom .x-tab-right { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/tabs/tab-btm-inactive-right-bg.gif) } .x-tab-strip-bottom .x-tab-left { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/tabs/tab-btm-inactive-left-bg.gif) } .x-tab-strip-bottom .x-tab-strip-active .x-tab-right { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/tabs/tab-btm-right-bg.gif) } .x-tab-strip-bottom .x-tab-strip-active .x-tab-left { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/tabs/tab-btm-left-bg.gif) } .x-tab-strip .x-tab-strip-closable A.x-tab-strip-close { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/tabs/tab-close.gif) } .x-tab-strip .x-tab-strip-closable A.x-tab-strip-close:hover { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/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(/core/ext.ncbi/1/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: = url(/core/ext.ncbi/1/img/oldentrez/tabs/scroll-right.gif); = BORDER-BOTTOM-COLOR: #aeaeae; BORDER-TOP-COLOR: #aeaeae; = BORDER-RIGHT-COLOR: #aeaeae } .x-window-proxy { BORDER-LEFT-COLOR: #b0b0b0; BACKGROUND: #e0e0e0; BORDER-BOTTOM-COLOR: = #b0b0b0; BORDER-TOP-COLOR: #b0b0b0; BORDER-RIGHT-COLOR: #b0b0b0 } .x-window-tl .x-window-header { COLOR: #555 } .x-window-tc { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/window/top-bottom.png) } .x-window-tl { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/window/left-corners.png) } .x-window-tr { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/window/right-corners.png) } .x-window-bc { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/window/top-bottom.png) } .x-window-bl { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/window/left-corners.png) } .x-window-br { BACKGROUND-IMAGE: = url(/core/ext.ncbi/1/img/oldentrez/window/right-corners.png) } .x-window-mc { 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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=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 reverseString (inStr)=0A= {=0A= var outStr =3D ''=0A= =0A= for (i =3D0; i <=3D inStr.length; i++)=0A= {=0A= outStr =3D inStr.charAt(i) + outStr=0A= }=0A= =0A= return outStr=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 reverseAndReplaceString (inStr, findStr, newStr)=0A= {=0A= return reverseString(inStr).replace(findStr, newStr)=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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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= =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= // elements "a", "area" with "ref" attribute report to redirect utility = before dereferencing link=0A= //=0A= function initRedirectClicks(report_url) {=0A= =0A= if (report_url && typeof jQuery !=3D 'undefined')=0A= jQuery(document).click(function(e) {=0A= =0A= //=0A= if (!e) return true;=0A= //=0A= if (e.shiftKey || e.altKey || e.ctrlKey) return true;=0A= //=0A= var target =3D jQuery(e.target).closest("a[ref], area[ref]");=0A= if (!target || target.length =3D=3D 0) return true;=0A= //=0A= var ref =3D target.attr("ref");=0A= var href =3D target.attr("href")=0A= =0A= // special processing of href element for pubmed's authors=0A= if (typeof (href) !=3D 'undefined' && = href.indexOf('sites/entrez') && href.indexOf('%5Bauth%5D'))=0A= {=0A= var newHref =3D replacePubMedUnsupportedChars(href);=0A= if (newHref !=3D href)=0A= target.attr("href", newHref)=0A= }=0A= =0A= // append ref with value of 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.$$handleEvent(event) =3D=3D=3D false) {=0A= returnValue =3D false;=0A= }=0A= }=0A= return returnValue;=0A= };=0A= =0A= function fixEvent(event) {=0A= // add W3C standard event methods=0A= event.preventDefault =3D fixEvent.preventDefault;=0A= event.stopPropagation =3D fixEvent.stopPropagation;=0A= return event;=0A= };=0A= =0A= fixEvent.preventDefault =3D function() {=0A= this.returnValue =3D false;=0A= };=0A= =0A= fixEvent.stopPropagation =3D function() {=0A= this.cancelBubble =3D true;=0A= };=0A= },=0A= =0A= // a counter used to create unique IDs=0A= addEvent_guid: 1,=0A= =0A= removeEvent: function(element, type, handler) {=0A= // delete the event handler from the hash table=0A= if (element.events && element.events[type]) {=0A= delete element.events[type][handler.$$guid];=0A= }=0A= }=0A= }=0A= =0A= =0A= // = *************************************************************************= ***=0A= // implementation of figure's popup using jQuery and = jQuery.fn.hoverIntent lib=0A= // it set appropriate handlers for all "a" elements with "figpopup" = class name.=0A= // = *************************************************************************= ***=0A= if (typeof jQuery !=3D 'undefined' && typeof jQuery.fn.hoverIntent !=3D = 'undefined') {=0A= (function($j) {=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 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=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_010F_01CA8591.191A8140 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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