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Malignant astrocytic brain tumors are among the most lethal cancers. Quiescent, and therapy-resistant neural stem cell (NSC)-like cells in astrocytomas are likely to contribute to poor outcome. Malignant oligodendroglial brain tumors, in contrast, are therapy-sensitive. Using magnetic resonance imaging (MRI) and detailed developmental analyses, we demonstrated that murine oligodendroglioma cells show characteristics of oligodendrocyte progenitor cells (OPCs), are therapy-sensitive; and that OPC rather than NSC markers enriched for tumor formation. MRI of human oligodendroglioma also suggested a white-matter (WM) origin, with markers for OPCs rather than NSCs similarly enriching for tumor formation. Our results suggest that oligodendroglioma cells show hallmarks of OPCs, and that a progenitor rather than a NSC origin underlies improved prognosis in patients with this tumor.
The relationship between oligodendroglioma cells and normal stem and progenitor cells is uncertain. Most normal brain structures arise from NSCs in the ventricular zone, an area that persists in adults and generates adult NSCs. Oligodendrocyte progenitor cells (OPCs) located in WM constitute a second postnatal reservoir for generation of glial cells. Whereas astrocytic tumors have been described to contain NSC-like tumor cells that are quiescent and therapy-resistant; the features of tumor-initiating cells in oligodendroglioma remain poorly understood. We show that mouse and human oligodendroglioma cells share hallmarks of progenitors rather than NSCs. Our results suggest that a progenitor origin for oligodendroglioma contribute to its responsiveness to therapy.
Oligodendrogliomas comprise a glial fibrillary acidic protein (GFAP) negative glioma, account for ~5–20% of gliomas, and show morphology and markers associated with oligodendrocytes, myelin-forming cells in the brain. Postnatal oligodendrocytes arise from oligodendrocyte progenitor cells (OPC), the most abundant population of cycling cells in the adult brain (Dawson et al., 2003; Geha et al., 2009). OPCs are widely dispersed in the subventricular zone (SVZ), a neural stem cell (NSC)-rich region lining the lateral ventricular walls, and as a resident population in white matter (WM) regions (Levison and Goldman, 1993; Menn et al., 2006; Zhu et al., 2008). OPCs can be identified through co-expression of platelet-derived growth factor receptor α (PDGFRα), transcription factors Sox10 and Olig2, and the neuro-glial chondroitin sulfate proteoglycan 4 (NG2) (Chang et al., 2000). Expression of NG2 is higher in oligodendrogliomas than in the more frequently arising astrocytic tumors, however lineage relationships among oligodendrogliomas, NSCs and OPCs remain poorly understood (Shoshan et al., 1999). In this communication, we investigated and compared NSCs and OPCs as potential cells of origin in murine and human oligodendroglioma.
To investigate oligodendroglioma development, we employed a transgenic mouse glioma model driven by an activated allele of EGFR, (v-erbB) under control of the human S100β promoter (Weiss et al., 2003). Aberrant epidermal growth factor receptor (EGFR) signaling in both NSCs and OPCs may contribute to oligodendrocytic tumors (Gonzalez-Perez et al., 2009; Ivkovic et al., 2008). S100β is associated with mature astrocytes, ependymal cells, select neuronal populations, and OPCs. In the adult SVZ, S100β is expressed as GFAP+ cells lose NSC potential (Hachem et al., 2005; Raponi et al., 2007). Mice expressing v-erbB develop low-grade oligodendrogliomas, with expression of v-erbB mRNA localized to the cerebellar granular cell layer, subcortical WM and SVZ (Weiss et al., 2003). Tumors arose with increased grade and shortened latency (average 66 ± 5d) in v-erbB-expressing mice deleted for p53 (E/p53−/−) and were used in most experiments (Figure S1A–B).
To assess proliferation in SVZ prior to development of tumors, we sacrificed mice at postnatal days 10 and 30 (P10 and P30). BrdU labeling in SVZ at P10 and P30 was indistinguishable in E/p53−/− mice and p53−/− littermates (Figure S1C–E). The distribution of BrdU in GFAP+ proliferating NSCs, doublecortin+ neuroblasts, and Olig2+ glial progenitors was also comparable in E/p53−/− and p53−/− mice (Figure S1F–H). These data suggest that v-erbB affected neither proliferation nor differentiation of SVZ NSCs, and are consistent with NSCs being S100β– (Raponi et al., 2007).
In contrast, tumor-bearing transgenic mice showed proliferation in stria terminalis, a WM structure adjacent to SVZ (Figure 1A–B, see insert). Also, irrespective of p53 status, symptomatic transgenic mice displayed massive proliferation in WM regions such as the corpus callosum (CC), illustrated by Ki67 or BrdU labeling (Figure 1C). To further localize tumors, we used MRI and postmortem histology in symptomatic transgenic mice. T1-weighted imaging of a transgenic animal illustrates a typical tumor within CC (Figure 1D). Tumor cells had characteristic oligodendroglioma-like morphology (Figure 1E–F), collectively suggesting that murine oligodendrogliomas arise in WM regions.
Glial progenitors, including NG2-expressing OPCs (Gonzalez-Perez et al., 2009; Menn et al., 2006), express high levels of Olig2, allowing us to distinguish these cells from NSCs and prompting us to quantify the fraction of Olig2+ glial progenitor cells that expressed NG2 in SVZ (Dawson et al., 2003). At P30, E/p53−/− mice showed a 3-fold increase in the fraction of Olig2+ cells expressing NG2 in SVZ compared to controls (Figure S1I–K). Further, all NG2+ cells co-expressed S100β (insert Figure S1J). These results suggest that OPCs expand in the SVZ of E/p53−/− mice. Although the SVZ is a region enriched for NSCs, our data are consistent with expression of v-erbB predominantly in OPCs rather than NSCs within the SVZ.
Since NG2+ OPCs are prominent in subcortical WM, we analyzed Olig2+ cells expressing NG2 in CC at P30. Transgenic mice (E/p53−/−) showed a 3-fold increase in the fraction of Olig2+ cells expressing NG2 in CC compared to controls (Figure 1G–I). Tumor regions arising in WM tracts of E/p53−/− mice expressed NG2 (Figure 1J–O). In cerebellum, most tumor cells were found in the innermost WM (Figure 1, panel O). At P30, E/p53−/− mice displayed high numbers of proliferative cells in other WM tracts (Figure 2A–D). These proliferative lesions stained for oligodendroglial markers NG2 and Olig2, and were negative for astrocytic marker GFAP (Figure 2E–J).
WM tracts in mice are enriched for OPCs, prompting us to investigate expression of additional OPC markers. NG2+ tumor cells co-expressed S100β, the v-erbB transgene, and the OPC markers PDGFRα, A2B5, Sox10, and Olig2 (Figure S2A). This expression profile together with expression of the oligodendrocyte markers MBP and CNPase, and low levels of GFAP are aligned with patterns in oligodendroglioma in humans. Collectively, these data suggest that tumors in this model arise from WM regions and express proteins normally found in OPCs.
To determine whether altered gene expression in transgenic OPCs contributed to tumors, we analyzed Sox10, a gene required for terminal differentiation of OPCs, that also promotes gliomagenesis (Johansson et al., 2004). We generated mice doubly transgenic for S100β-v-erbB and NG2-DsRed (Zhu et al., 2008), allowing us to identify and isolate DsRed+ NG2 cells. WM regions from E/p53−/− mice (NG2-DsRed) were pooled at P30, well before tumors arise. Tissues were micro-dissected and analyzed by FACS, followed by RNA isolation and real-time PCR (Figure S2B). Levels of Sox10 in NG2+ cells were 5-fold higher in E/p53−/− than in p53−/− mice (P < 0.05), suggesting that the S100β-v-erbB transgene regulates gene expression in NG2+ cells, promoting tumorigenesis (Figure S2C). These data demonstrate that expansion of OPCs in WM regions precedes development of tumors, and suggest that oligodendrogliomas in these animals arise from NG2+ cells in WM regions.
It is important to clarify that TICs define a functional population of cells that are tumor propagating, and does not necessarily imply a relationship with a specific cell of origin. TICs in human GBM typically constitute a minority population, and share features of NSCs including self-renewal, multipotency and resistance to therapy (Bao et al., 2006; Bleau et al., 2009; Singh et al., 2004; Stiles and Rowitch, 2008). To study these properties, we isolated cells from SVZ, WM tracts, and tumors of both E/p53−/− mice and p53−/− controls. Cells in Neurobasal media (NB) supplemented with FGF-2 and EGF were studied as acutely isolated, low-passage (< 5 passages), or high-passage (>5 passages).
To delineate the relationship among v-erbB expressing cells isolated from SVZ and tumors (E/p53−/−), normal NSCs from SVZ (p53−/−), and WM progenitors (p53−/−), we performed real-time PCR for NSC and OPCs markers. Transgenic (E/p53−/−) cells from SVZ and tumors expressed high levels of OPC markers and low levels of NSC markers relative to normal SVZ NSCs (Figure S2D). Interestingly, v-erbB-expressing low-passage cells isolated from SVZ, CC, and tumors showed gene expression profiles similar to control cells isolated from CC rather than SVZ (Figure S2E). In fact, levels of OPC markers Sox10 and PDGFRα were elevated in acutely and low-passage cells from E/p53−/− mice compared to WM progenitors from p53−/− mice, suggesting that v-erbB was associated with increased expression of these genes (Figure S2E–F).
In contrast to passaged SVZ cells from E/p53−/− mice (Figure S2E), acutely isolated SVZ cells expressing v-erbB mRNA showed no changes in Sox10 and Hes1 compared to controls (Figure S2F). We confirmed for a subset of these genes, differential protein expression in E/p53−/− tumor cells (10118T) compared to p53−/− SVZ NSCs (Figure S2G). The high expression levels of OPC rather than NSC markers in v-erbB-expressing cells suggest that these cells are more related to WM OPCs rather than SVZ NSCs.
In contrast to SVZ NSCs, murine oligodendroglioma cells showed efficient proliferation independently of growth factors (Figure 3A, S3A–B). Basal levels of pErk1/2 and pAkt (in SVZ NSCs, grown in FGF-2 and EGF) were reduced when compared to low-passage E/p53−/− cells (Figure 3B). We next analyzed proliferation in NG2+ OPCs. Dividing BrdU+ low-passage cells in SVZ NSC cultures from p53−/− mice were NG2−. In contrast proliferating tumor cells were highly NG2+ (Figure S3C–D). NG2+ cells from transgenic mice expressed both S100β and v-erbB (Figure S3E–H). The frequency of NG2+ cells in SVZ cultures from E/p53−/− mice was expanded in and adjacent to SVZ, with unaltered proliferation of SVZ NSCs (Figure S3I–J). These results suggest that v-erbB under control of the S100β promoter promotes expansion of NG2+ OPCs.
To modulate signaling pathways, we used MEK inhibitor PD325901 (1 μM, PD) and PI3K/mTOR inhibitor LY294002 (10 μM, LY). Tumor cells from transgenic (E/p53−/−) mice were incubated without EGF and FGF-2 for 24h. Inhibitors were added (2h) followed by 100 ng/ml EGF (20min) prior to lysis (Figure S3K). To determine effects on low-passage v-erbB-expressing murine oligodendroglioma (E/p53−/−), we incubated cells for 7d with PD (1 μM) and/or LY (10 μM). While PD was more effective than LY in reducing proliferation of SVZ NSC (p53−/−SVZ), both inhibitors were equally effective at reducing proliferation of tumor cells (Figure S3L). Interestingly, inhibition of MEK (but not PI3K/mTOR) induced oligodendrocyte-like differentiation of the tumor cells (Figure S3M–N).
Incubation with PD differentiated acutely isolated murine oligodendroglioma (31194T) cells into oligodendrocytes expressing O4, a marker of immature oligodendrocytes (Figure 3C). PD reduced the percentage of NG2+ tumor cells (67 ± 4% vs. 21 ± 5%, P < 0.001) and increased the percentage of O4+ cells (11 ± 3% vs. 51 ± 3%, P < 0.01) (Figure 3D), specifically inducing oligodendroglial, but not neuronal or astroglial, differentiation. Incubation of SVZ NSCs with PD induced neuronal (map2ab: 32 ± 4% vs. 3 ± 2%, P < 0.001) and astroglial (GFAP: 29 ± 2% vs. 5 ± 1%, P < 0.001), but not oligodendroglial differentiation (Figure 3E–F). These results suggest that the S100β promoter drives expression of v-erbB expression in WM OPCs, resulting in increased pErk1/2, increased proliferation, and formation of oligodendroglioma in transgenic mice.
We next investigated E/p53−/− oligodendroglioma cells for multipotency and sphere-formation. Both ciliary neurotrophic factor (CNTF) and bone-morphogenic protein 4 (BMP4) induce robust astroglial differentiation in low-passage NSC cultures. Incubation with these factors for 7d reduced the expansion of low-passage SVZ NSCs and astrocytoma (GFAP-Ras) cells (Ding et al., 2001), with more modest effects on low-passage oligodendroglioma cells (Figure 4A–C), suggesting that oligodendroglioma cells fail to respond to morphogens that block proliferation in both NSCs and astrocytoma cells.
To further assess the multilineage competence of oligodendroglioma-derived cells, we treated cells with morphogens known to differentiate NSCs into neurons (forskolin), astrocytes (CNTF or BMP4) and oligodendrocytes (combination of T3 and IGF-1), analyzing both acutely isolated SVZ NSCs (p53−/−) and tumor cells (31194T). Consistent with a limited differentiation potential of OPCs, NG2-expressing tumor cells responded to forskolin by differentiating into O4+ oligodendrocytes rather than neurons or astrocytes (Figure 4D–E). While BMP4 showed an inhibitory effect on oligodendrocyte differentiation, neither CNTF nor T3/IGF1 significantly affected differentiation in oligodendroglioma cells (Figure 4D–E).
In contrast to the limited differentiation potential of E/p53−/− oligodendroglioma cells, p53−/− SVZ NSCs were multipotent, and showed robust differentiation into neurons (forskolin), astrocytes (CNTF or BMP4), and oligodendrocytes (T3/IGF-1) (Figure 4D,F). In tumors from a murine astrocytoma model (GFAP-Ras), acutely isolated tumor cells expressed CD15 and readily differentiated into astrocytes in response to forskolin, CNTF, or BMP4 (Figure S4A). Furthermore, continued passage of these astrocytoma cells enriched for the NSC marker CD15 and not the OPC marker NG2 (not shown). Thus, NSCs and murine astrocytoma cells both show multipotent differentiation potential, while differentiation potential in murine oligodendroglioma cells is more restricted.
To determine if the restricted differentiation of murine oligodendroglioma cells was differentially associated with either NG2+ or NG2− subpopulations, we acutely isolated tumor cells, and separated NG2+ and NG2− populations using FACS. One day after isolation many NG2− cells displayed an oligodendrocyte phenotype, whereas NG2+ cells remained undifferentiated morphologically, continuing to divide (Figure S4B). Enzymatic dissociation of murine oligodendroglioma cells cleaved the NG2 epitope, less with accutase than with trypsin (Figure S4C and not shown). Immediately after accutase dissociation, cells analyzed by FACS showed a pure NG2− cell fraction (Figure S4C). Incubation of NG2− and NG2+ oligodendroglioma cells for 7d with forskolin revealed that NG2+, but not NG2− cells, could differentiate into O4+ oligodendrocytes (Figure S4D–E). Few tumor cells (9 ± 4%) in NG2-sorted cultures expressed neuronal marker map2ab. In contrast to NSC cultures (p53−/−SVZ), this number was unchanged in response to forskolin. These results suggest that NG2+ cells can be differentiated along the oligodendrocytic lineage. We found that NG2+ oligodendroglioma cells in this mouse model show poor sphere-formation capacity, and that CD15 labels a population of sphere-forming cells in both tumors and the non-transformed brain (Figure S4F–G). To study how passage affected the fraction of cells expressing NG2 or the NSC marker CD15, we cultured tumors from transgenic mice and performed FACS at different passages. Continued passage enriched for NG2+ cells due to death of non-tumor cells and fast proliferation of NG2+ tumor cells (Figure S4H). Expression of CD15 did not change (Figure S4I).
In murine astrocytoma models, sphere-forming tumor cells effectively establish tumors in-vivo (Alcantara Llaguno et al., 2009; Marumoto et al., 2009), with the NSC marker CD15 enriching for sphere-forming TICs in human GBM (Son et al., 2009). Consistent with a progenitor rather than an NSC-origin in murine oligodendrolioma, cells isolated from tumors of E/p53−/− mice displayed a major population of NG2+ cells that was non-overlapping with a minor CD15+ population (Figure 5A). To investigate whether CD15+ cells could initiate oligodendroglioma, we grafted 1,000 low-passage CD15− (CD15 neg) or CD15+ (CD15 high) tumor cells into recipient mice. CD15+ NSC-like cells isolated from E/p53−/− mice were non-tumorigenic (Figure 5B). In contrast, grafting of 50 acutely isolated NG2+CD15− tumor cells resulted in tumors in 3/4 mice (Figure 5C). Control mice injected with NG2−CD15− or NG2−CD15+ cells showed no tumors over 60d.
Since continued passage of tissues from E/p53−/− mice enriched for NG2 expression (Figure 5D), we used low-passage cells, sorted these into NG2 low, intermediate or high groups, and grafted 1,000 tumor cells into recipient mice. Expression of NG2 correlated with tumorigenicity (Figure 5E). To further define the efficiency by which NG2-directed FACS enriched for TICs, we grafted 50 NG2+ or NG2− tumor cells from acutely, low- and high-passaged tumors into recipient mice. Independently of passage, NG2+ cells were more tumorigenic than NG2− cells (Figure 5F). All tumors showed oligodendroglioma-like morphology, with transplanted and primary tumors showing the same OPC markers (data not shown).
A dye-excluding minority side-population (SP) of tumor cells also shows properties of NSCs (Murayama et al., 2002). To investigate the SP in murine oligodendroglioma, we FACS sorted tumor cells from E/p53−/− mice and compared tumorigenicity in SP and non-SP (NSP) tumor cells injected into recipient mice. The SP (0.9 ± 0.7% of all tumor cells) was abolished by incubation with 20 μM verapamil, known to block SP-enriched multidrug transporters (Figure S5A). No significant difference in tumorigenicity was observed between SP and NSP cells (Figure S5B). Both the SP and S100A6 have been suggested to mark rare NSC-like tumor cells in this model (Harris et al., 2008). We observed broad expression of S100A6 in all tumors (Figure S5C). Our results suggest that neither the SP nor S100A6 enrich for TICs in this model.
The cell-surface marker CD133 also enriches for TICs in human glioma (Singh et al., 2004). The corresponding murine protein prominin, was expressed at low levels in v-erbB+ tumors (5 of 6 tumors showed <1% prominin+ cells), and overlapped with both the NG2− and NG2+ populations (Figure S5D–E). The latency of tumor formation in recipient mice and ability to form tumor spheres in vitro reflected the fraction of NG2+ cells in both prominin− and prominin+ fractions (Figure S5F–H). In a separate experiment, few CD15+ NSCs (p53−/−SVZ) incorporated BrdU after a 24h pulse whereas the majority of NG2+ oligodendroglioma cells (E/p53−/−) were labeled (Figure S5I–L). Collectively, these results demonstrate that NG2 labels a major population of fast-dividing tumor cells displaying higher tumorigenicity than NG2− cells in murine oligodendroglioma.
To correlate the OPC origin with response to alkylator therapy in oligodendroglioma, we incubated SVZ NSCs, murine oligodendroglioma cells, and murine astrocytoma (GFAP-Ras) cells with TMZ (TMZ, 10–100 μM). In contrast to SVZ NSC and murine astrocytomas (Figure S6A–B), murine oligodendrogliomas and OPCs (p53−/−CC) were highly temozolomide-sensitive (Figure 6A–E, S6C). Treatment of NG2− and NG2+ fractions showed both populations to have equivalent sensitivity to TMZ (Figure 6F). These data suggest further that OPC rather than NSC-like cells in oligodendroglioma correlate with improved response to alkylating agents.
To determine relevance, we isolated human oligodendroglioma cells from a patient with a grade II oligodendroglioma (SF8245). As a comparison, we also isolated tumor cells from a patient diagnosed with a grade II astrocytoma associated with the lateral ventricles (SF8138), and studied low-passage primary human GBM cells (SF6969). When low-passage human tumor cells were cultured in neurobasal media (NBE, see Supplemental Experimental Procedures) on coated plates and incubated for 7d with 10–100 μM TMZ, oligodendroglioma cells proved more sensitive than either astrocytoma or primary GBM cells (Figures 6G, S6D–E). Interestingly both human GBM and astrocytoma cultures showed enrichment of CD15+ cells after treatment with TMZ, with the GBM also demonstrating higher levels of CD133 and podoplanin (not shown). Like murine oligodendroglioma cells, human oligodendroglioma cells, irrespective of NG2 status, were similarly sensitive to TMZ (Figure 6H). These human and murine data collectively suggest that a progenitor-like phenotype may underlie sensitivity of oligodendroglioma cells to TMZ, and that NSC-like properties reciprocally underlie relative resistance to TMZ in astrocytic tumors.
To study tumorigenicity of NG2+ cells in human glioma, we dissociated tumor cells, grown in NBE supplemented with FGF-2 and EGF, from 2 patients diagnosed with primary grade II oligodendroglioma (SF7987 and SF7891). Flow cytometry verified expression of NG2 in a significant fraction of all samples (Figure 6I–J). Interestingly, continued passage in vitro of human oligodendroglioma cells (SF8245) cultured in NBE supplemented with FGF-2 and EGF, led to further enrichment for NG2, while similar propagation of primary human GBM enriched for CD15 (data not shown).
To study tumorigenicity, we fractionated acutely isolated SF7987 oligodendroglioma cells followed by grafting of 1,000 NG2+ or NG2− cells. Three of 4 mice injected with NG2+ oligodendroglioma cells developed tumors after 5–8 months. No tumors (0/4) established from NG2− SF7987 cells (Figure 6K). Two mice injected with 1,000 acutely isolated NG2+ low grade oligodendroglioma cells SF7891 (2/4), died after almost 1 year, whereas all mice injected with NG2− cells survived (Figure 6L). These results suggest that NG2+ cells from human oligodendrogliomas proliferate and are highly tumorigenic.
If oligodendrogliomas arise from OPCs rather than NSC-like cells, then do these tumors associate with white-matter regions where NG2+ OPCs normally reside? To address this question, we studied 47 grade II and III non-enhancing oligodendrogliomas and astrocytomas. Use of small lower grade tumors simplified localization to WM or ventricular regions. Further, the fact that tumors did not exhibit contrast-enhancement reduced the chances of erroneously identifying the boundaries of leaky vasculature as the boundaries of tumor infiltration.
Using T1-weighted spoiled gradient echo (SPGR) and T2-weighted fast low angle inversion recovery (FLAIR) MRI images, we determined whether 47 grade II–III gliomas were touching the lateral ventricles (Figure 7A). We hypothesized that gliomas touching the lateral ventricles were astrocytomas (Figure 7B). For the remaining gliomas, the diagnosis was predicted using FA and apparent diffusion coefficient (ADC) maps, highlighting regions of fluid movements (Khayal et al., 2009). We first performed a qualitative assement of the FA map to classify as oligodendroglioma (diffuse edges) or astrocytoma (well-circumscribed borders) and then confirmed the classification by calculating the median normalized ADC value (Figure 7C). The qualitative and quantitative assessments matched in 18/20 cases that did not touch the ventricles. When they did not match, the ADC value was used because of the previously reported predictive value (Khayal et al., 2009). The majority of astrocytomas (Type 1: 21/34) were touching the lateral ventricles (Figure 7D). Based on ADC values, we were able to distinguish between 5 astrocytomas (Type 2, ADC: 2.1 ± 0.1) and oligodendrogliomas (ADC: 1.5 ± 0.02 located), not touching the ventricles. A diagnosis for a third group of 8 astrocytomas (Type 3) could not be predicted based on location or ADC values. All human oligodendrogliomas showing loss of chromosomes 1p and 19q (11/11), were associated with WM tracts rather than lateral ventricles (Figure 7E), consistent with an origin from WM progenitor cells. These results suggest that localization of gliomas and diffusion pattern on MRI may be useful future tools to subclassify oligodendrogliomas and astrocytomas with an OPC− or NSC−signature.
Based on these data, we predicted that progenitor cells within human oligodendroglioma would express transcripts consistent with human WM glial progenitors (here denoted OPCs), rather than with that of ventricular zone NSCs. To test this, we used magnetic cell immunosorting using an antibody against A2B5, which recognizes glial progenitor cells. A2B5 is expressed in the majority of tumor cells in oligodendroglioma (Ogden et al., 2008), and is typically co-expressed with NG2 (Sim et al., 2006). The relative abundance of a set of NSC− and OPC-related genes was compared in A2B5+ cells isolated from grade II human oligodendrogliomas (n=3), astrocytomas (n=4), and human OPCs, and normalized to their depleted (A2B5−) remainder. In a set of 91 cell-type and pathway-specific genes, 15% and 34% of the studied genes in oligodendroglioma and astrocytoma, respectively, showed significantly (P < 0.05) different mRNA expression levels compared to human OPCs (Figure 7F). Astrocytomas expressed lower levels of the OPC-related genes CSPG4 (NG2), Nkx2.2, and PDGFRα, compared to OPCs. Similar levels of the OPC-related genes (NG2, Olig2, Nkx2.2, PDGFRα, and Sox11) and NSC-related genes (Nestin, Sox2, Sox9, Musashi1/2) in human oligodendroglioma cells and OPCs suggest a progenitor rather than a stem-cell origin.
We next investigated sphere-formation and multipotency in human oligodendroglioma. Human tumors were acutely isolated and cultured in N5 media, NBE media, or M41 media supplement with N1 and FGF2 (see Supplemental Experimental Procedures). An oligodendroglioma isolated from frontal cortex grew as elongated cells on coated plates in NBE (SF8245, Figure 8A–B). Incubation in M41 media resulted in slower growth along with differentiation into cells showing oligodendroglial morphology (inset Figure 8B). Some oligodendrogliomas expanded in NBE grew as clusters rather than uniform spheres on ultra-low adherent plates (Figure 8C).
To evaluate the ability to form spheres, we plated cells from three low-passage grade IV astrocytomas (primary GBMs), an acutely isolated grade II astrocytoma (SF8138), and 2 acutely isolated grade II oligodendrogliomas in NBE (Figure 8D). In contrast to unsorted, NG2+, and NG2− oligodendroglioma cells, 4 astrocytic tumors efficiently formed spheres. These results suggest that human oligodendrogliomas are deficient at clonogenic expansion, unlike either primary NSCs or cells derived from astrocytomas or GBMs.
We assayed proliferation and differentiation potential by incubating human oligodendroglioma culture SF8245 for 2h with BrdU. Approximately 10% of NG2+ cells incorporated BrdU and expressed the glial progenitor marker Nkx2.2 (Figure 8E). The NG2+ cells expressed nestin (Figure 8F) but at lower levels than GBM cells (Figure S7A–C). We next incubated human oligodendroglioma cells (passaged once) with forskolin, BMP4, and T3/IGF to induce differentiation along the three neural cell lineages. Similar to murine oligodendrogliomas, neither BMP4 nor a combination of T3 and IGF1 induced differentiation in this human oligodendroglioma (Figure 8G). Also similar to the transgenic mouse model, incubation with forskolin reduced the levels of NG2+ oligodendroglioma cells (79 ± 6% vs 33 ± 6%, P < 0.01) and increased the levels of O4+ oligodendrocyte-like cells (0 ± 0% vs 7 ± 0.6%, P < 0.001) (Figure 8G–I).
A significant fraction (13 ± 1%) of the tumor cells expressed the immature neuronal marker TujI, suggesting ability to differentiate into neurons. However, forskolin did not increase neuronal differentiation of human oligodendroglioma cells. Incubation of grade II human oligodendroglioma cells (SF8245) with the MEK inhibitor PD325901 (1 μM) similarly reduced the fraction of NG2+ cells (79 ± 6% vs 32 ± 4%, P < 0.01) and induced expression of O4+ oligodendrocyte-like cells (0 ± 0% vs 8 ± 2%, P < 0.01) (Figure S7D–E). These results are aligned with those obtained using murine v-erbB-expressing oligodendrogliomas, and suggest that human oligodendroglioma cells are not tripotent, mainly differentiating into oligodendrocyte-like cells.
Recent studies suggest a relationship between NSCs and their malignant counterparts. However it is equally tenable that in the course of malignant progression, restricted progenitors or even differentiated cells may acquire properties associated with TICs: self-renewal, multipotent differentiation, and asymmetric division. It is entirely possible that tumor cells with a progenitor-like phenotype could arise from stem cells that subsequently commit to a progenitor lineage (e.g. BCR-Abl mutations in stem cells giving rise to myeloid leukemia (Castor et al., 2005)), or from a progenitor that subsequently de-differentiates into a more stem-like cell (Akala et al., 2008). Given these caveats, it is important to distinguish between the normal cells from which the tumor arises, and the cells that are capable of propagating tumors following transplantation.
Unlike NSCs, OPCs undergo a restricted number of cell divisions before they differentiate, a timing mechanism partly controlled by p27Kip1 (Durand et al., 1997). Since expression of v-erbB reduces p27Kip1, and proliferation in human oligodendrogliomas is inversely correlated with p27Kip1 levels (Fan et al., 2003; Fiano et al., 2003), this timing mechanism might be dysregulated in both murine and human oligodendroglioma cells, allowing tumor cells to continuously self-renew and unable to differentiate.
OPCs immortalized with an activated, point-mutated erbB2 oncogene differentiate into immature and mature oligodendrocytes in response to forskolin or erbB2 inhibition (Gobert et al., 2009). Similar to these OPCs, NG2-expressing oligodendroglioma cells in our studies responded to forskolin or a MEK inhibitor by differentiating into O4+ oligodendrocytes (and not into astroglia or neurons), whereas BMP4 treatment inhibited oligodendroglial differentiation.
Nuclear export of Olig2 is essential for complex formation between STAT3 and the transcription activator p300, allowing CNTF-induced differentiation in NSCs and OPCs (Setoguchi and Kondo, 2004). High nuclear expression of Olig2 in NG2+ oligodendroglioma cells might therefore explain why these cells failed to differentiate in response to CNTF. Interestingly, some NG2-sorted tumor cells expressed the neuronal marker map2ab, suggesting that these cells did differentiate into neurons. Similar to the v-erbB driven murine oligodendrogliomas, cultures of a 1p/19q deleted grade II oligodendroglioma (SF8245) contained a subpopulation of cells expressing the neuronal marker TujI along with oligodendroglial differentiation in response to forskolin, T3/IGF1, or a MEK inhibitor. Collectively, these data suggest that murine oligodendroglioma cells show a gene expression profile consistent with OPCs, and are bipotent rather than multipotent, differentiating mainly into oligodendrocytes.
Our previous study in mice transgenic for S100β-v-erbB showed tumors to arise in supratentorial and hindbrain locations (Weiss et al., 2003). Mutation at p53 led to increased grade and penetrance, while also promoting a more supratentorial localization. Perhaps relevant to these data, 13% of oligodendrogliomas and 25% of high-grade gliomas show p53 mutations, whereas mutation in p53 are rare in medulloblastoma, a tumor of the posterior fossa (Adesina et al., 1994). This caudal-rostral shift of tumors suggests that p53 plays region-specific roles in regulating proliferation, differentiation, and senescence in NSCs and progenitors during brain development that influences localization of tumors (Marumoto et al., 2009). We cannot exclude an NSC or radial glial origin for tumors arising in the posterior fossa region. However, S100β is expressed in embryonic radial glia lining the 4th ventricle (Hachem et al., 2007), and from birth is mainly localized to glia in the cerebellar WM (Vives et al., 2003), the region where we observed most NG2+ oligodendroglioma cells.
Loss of chromosomes 1p and 19q occurs commonly in human oligodendroglioma, especially in proneural tumors, while over-expression of EGFR and loss of p53 mark only a subset of tumors (Ducray et al., 2008). Interestingly, a minority of tumors in the v-erbB model shows loss of the distal part of chromosome 4 that corresponds to human 1p (Weiss et al., 2003). We demonstrate that OPC-like human oligodendroglioma cells from both 1p/19q and EGFR-driven tumors were highly tumorigenic. MRI analyses from patients with grade II–III gliomas demonstrated that oligodendrogliomas and astrocytomas could be subclassified based on MRI diffusion patterns (Khayal et al., 2009) and association with the lateral ventricles, a NSC-rich region. Interestingly, all 1p/19q deleted tumors arose in WM regions, while two EGFR-driven p53 mutant human tumors were associated with the lateral ventricles.
Despite the association of different tumors with specific brain regions, the cell of origin in human glioma remains uncertain. Recent subclassifications have demonstrated a classical group of primary GBMs with high levels of the NSC marker nestin and EGFR (Verhaak et al., 2010), and a mesenchymal group controlled by a transcriptional network that transforms NSCs (Carro et al., 2010). These two subclasses of primary GBMs may thus arise from transformation of NSCs. A proneural group of GBMs displays mutation of IDH1 and shows high levels of the OPC-related genes Nkx2.2, Olig2, and PDGFRα (Brennan et al., 2009; Verhaak et al., 2010). In contrast to other groups of GBMs, the gene expression profile of proneural GBMs was aligned with cultured oligodendrocytes (Verhaak et al., 2010), suggesting that IDH1 mutant gliomas may arise from OPCs rather than dedifferentiated astrocytes or NSCs. Proneural oligodendrogliomas also show 1p/19q loss, and express neuronal markers consistent with origin from a bi-potential progenitor capable of generating both oligodendrocytes and neurons (Ducray et al., 2008).
The NSC markers CD133 (prominin), CD15, and the dye-excluding SP enrich for highly tumorigenic cells in the brain tumors medulloblastoma and astrocytomas, in both mice and humans (Bleau et al., 2009; Marumoto et al., 2009; Read et al., 2009; Singh et al., 2004; Son et al., 2009). In our studies, isolated CD15+ cells from murine oligodendroglioma, were unable to form tumors, whereas tumorigenicity of rare CD133+ tumor cells could be explained by partial overlap with the OPC marker NG2. The discrepancy in tumorigenicity between SP and NSP cells between our study and a previous report could reflect NG2+ cells in the NSP (Harris et al., 2008). Higher levels of NG2, PDGFRα, Nkx2.2, Sox2, and S100β were reported in the SP compared to the NSP, suggesting that the NSP contained fewer NG2+ tumor cells (Harris et al., 2008). We observed NG2+ cells in both SP and NSP, perhaps explaining the similar latency between these groups in our experiments.
In contrast to results using NSC-based markers, we found that NG2+ enriched for tumor formation in vivo, in both murine and human oligodendroglioma. Our data suggest that under these conditions, NG2+ cells showed significantly higher (but not exclusive) tumorigenicity compared to NG2− oligodendroglioma cells. Enzymatic treatment with accutase and/or differentiation of NG2−CD15+ cells into NG2+ cells, could contribute to the abundance of NG2+ cells, that accumulated over time in cultured NG2− fractions, and might explain why grafting large numbers of NG2− cells led to tumors in recipient mice.
A transcriptional network in gliomas similar to either NSCs or OPCs, along with distinct chromosomal changes, may underlie differential response to alkylating agents and other therapies. We observed that murine SVZ NSC and human astrocytoma cultures were relatively resistant to treatment with TMZ. In contrast, murine OPCs and OPC-like tumor cells from transgenic mice and human oligodendrogliomas were highly sensitive. These results suggest that a progenitor origin contributes to the relative therapy-sensitive nature of oligodendroglioma. Parenchymal localization, MRI data, and immunohistochemical markers may help to select the appropriate glioma-selecte therapy, in conjunction with an OPC− or NSC-signature related to specific chromosomal aberrations.
Collectively, our data suggest that murine oligodendroglioma arise from NG2-expressing WM OPCs rather than NSCs. We demonstrate that NG2+ oligodendroglioma cells express genes and proteins associated with OPCs rather than NSCs, that NG2+ oligodendroglioma cells show limited sphere-formation, consistent with a progenitor population, and that NG2+ oligodendroglioma cells are lineage restricted. NG2-expressing cells from both mouse and human oligodendroglioma displayed high tumorigenicity and respond to both alkylating and differentiating agents. We show that these oligodendroglioma cells are neither chemo-resistant nor quiescent and suggest that a progenitor origin for these cells underlies their chemosensitivity. Our results suggest that oligodendroglioma arises from NG2-expressing cells in WM regions, and links the therapy-responsive nature of this tumor to a progenitor origin.
Mice transgenic for S100β-verbB (Weiss et al., 2003) and mice deleted for p53 (Donehower et al., 1992) (E/p53−/−, p53−/−) were maintained in a pure FVB/N background. Mice carrying the GFAP-Ha-V12-Ras transgene were maintained in a CD1 background. Genotypes were confirmed using PCR (Supplemental Experimental Procedures). All experimental procedures involving animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee at UCSF.
Mouse cells were isolated from brain regions using a stereo-dissection microscope (Leica). Tissues were dissociated using papain. Murine cells were cultured in neurobasal (NB) media (-A, Invitrogen) supplemented with 1xB27 supplement, 20 ng/ml FGF-2 (Peprotech), 20 ng/ml EGF (Sigma-Aldrich), 2 mM L-glutamine, and incubated at 37°C in 5% CO2. Human cells were used after review and approval from the committee on human research at UCSF and University of Rochester. Informed consent was obtained from all subjects. For all assays, human glioma cells were cultured in NBE media that is similar to NB (containing 0.5xB27 plus 0.5xN2 supplement (Gibco) rather than 1xB27 supplement alone). Cell culture based assays are described in Supplemental Experimental Procedures.
To detect proliferating cells in mice, BrdU (Sigma-Aldrich) was injected IP at 2 h before sacrifice (50 mg/kg). Paraffin-embedded (5 μm) and fixed (4% para-formaldehyde) free-floating (30 μm) sections were used for light microscopy and fluorescent stainings. See Supplemental Experimental Procedures.
For details on quantitative RT-PCR see Supplemental Experimental Procedures.
FACS-sorted human glioma cells (1,000–10,000 cells/mouse) or FACS-separated mouse tumor cells (50–1,000 cells) isolated from E/p53−/− mice were intracranially grafted into anaesthetized (Ketamine and methamidine) recipient NOD-SCID and FVB/N mice.
Statistical analyzes for all experiments including more than two groups were performed using a two-way ANOVA followed by posthoc comparisons using Dunnett's posthoc test. For experiments with two groups, we used Student's t-test.
We acknowledge Abhijit Guha for GFAP-Ras mice, and Michael Burns, Michael Wendland, and Tim Roberts for images. Supported by the Swedish Society for Medical Research, Swedish Medical Research Council, Hjarnfonden, Sandler Postdoctoral Fellowship, and Joel A. Gingras Jr/American Brain Tumor Association Fellowship (AIP); The Sandler Opportunity Award in Basic Science (GB); NIH SPORE CA095103 and R01 GM081635 (EL); The Children's Brain Tumor Foundation, NIH SPORE CA097257, The Brain Tumor Society, UCSF Academic Senate, Pediatric Brain Tumor, Alex's Lemonade Stand and Samuel Waxman Cancer Research Foundations, Accelerate Brain Cancer Cure and the Burroughs Wellcome Fund (WAW).
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Joy | Sep 5th 2006
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Stephanie Zom B Model
DD StudioPhotographer Male Warrington, Pennsylvania, US
My MM URL: http://www.modelmayhem.com/digitaldimensionsstudio
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About Me
Thank you for visiting my little corner of Model Mayhem. Up until now I have mainly been into landscape and nature photography. I just set up a studio and currently looking to enhance my portfolio glamour, portraits, fashion, and lingerie. I am always available for TFP/TFCD and will occasionally hire models from time to time. I prefer casual sessions and strive for conditions where everyone feels secure creative and collaborative. Please email me if you are looking to expand your portfolio.
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Articles Tagged Defensive Shift
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June 6, 2012 5:00 am
The defensive shift revolution makes for a nice narrative, but how much has it truly changed the game?
Last week, we examined the effects of fielding shifts on fielding metrics. For those who missed out, I'd advise you to go read it, but the short version is that location-based fielding metrics can overstate the importance of fielding shifts to a team's defense and thus overrate players who are shifted in such an arrangement.
But if the fielding shifts are throwing defensive metrics off, that doesn't mean that they aren't contributing to team defense, right? And we are in what some people might term a shifting renaissance. John Dewan of Baseball Info Solutions says:
June 21, 2010 9:00 am
These days, it seems like you can't discuss defensive shifts without a J.D.
Sometimes, it's the comments that seem to miss the mark that generate the most discussion. Take this nugget (please!) from Tom Verducci, buried within a long, otherwise-insightful article for SI.com about this season's league-wide ebb in offense:
"The state of hitting is awful. Batters strike out more than ever before and, anecdotally, there seem to be more defensive overshifts employed than ever before. A shift for J.D. Drew, a guy who has hit 30 homers once in his life?"
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January 10, 2008 12:00 am
Repositioning the infield and its effects on history, defense, and batting.
When asked what he did in the winter when there is no baseball, Rogers Hornsby famously replied that he would "stare out the window and wait for spring." Fortunately, these days fans of the game have plenty to keep them occupied in the cold winter months. Not least amongst the off-season events is looking forward to the baseball-oriented Christmas loot that keeps us busy until spring. While not the massive haul of our Jay Jaffe, I'll keep warm with a new Cubs fleece from my lovely wife and keep occupied with Lee Lowenfish's hefty biography of Branch Rickey titled Branch Rickey: Baseball's Ferocious Gentleman and Cait Murphy's romp through the 1908 season Crazy '08: How a Cast of Cranks, Rogues, Boneheads, and Magnates Created the Greatest Year in Baseball History; both additions to my library courtesy of my in-laws.
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With such big-budget TV blockbusters as The Pacific, Boardwalk Empire and The Walking Dead on our screens and in our DVD collections, it should come as no surprise that much of our free time this year has been spent not only watching the original Star Wars trilogy over and over (hey, it's traditional around here), but also the cream of American and British TV. Looking back over the past 12 months, here's our brief glance over the best the little box in the corner of your living room has had to offer…
When HBO commissioned this $200m mini-series, the first, obvious question that popped into people's minds was: "Will it be as good as Band Of Brothers?" Followed by, "HOW much?!" It was true that the shadow of Winters, Guarnere and Easy Company hung heavily over this second slab of WW2 drama, but The Pacific definitively lived up to expectations.
A very different animal from Brothers, a broader ensemble piece set in the European theatre, The Pacific centred on three Marines struggling to survive the hellish island campaign with their humanity intact - as well as a fourth, Snafu, who already seemed to have lost his. The journeys of the men, real-life veterans Leckie, Sledge and Basilone, was movingly portrayed. No shelter was taken in mawkish cliché or over-reverence.
These were flawed men just trying to get through, it said; "how would you cope?" the unspoken question. Surprisingly, despite performances from its leads that lent the three storylines quiet power, the acting was overlooked at the Emmys. And what of that $200m? Well, that did hold sway with the Emmy judges, who made it their Best Miniseries. Watching the ten, hour-long episodes, at times it looked as if they actually did recreate the entire war. Spectacular, violent and terrifying, the battle scenes billowed cordite from our tellys. Get hold of the discs if you missed the broadcast.
With humour so black you could stick legs on it and call it a badger, AMC's mordantly brilliant, macabre, at times plain-ass weird miniseries went from strength to strength this year. Its third season took chemistry-teacher-turned-meth-kingpin Walter White (Bryan Cranston) deeper into the rabbit warren of supply and demand, black and white, and all shades in between.
Cranston was again terrific as an occasionally befuddled, permanently embattled man who's given lemons and decides to synthesize them into high-grade Class-A lemonade. Cranston was again the Emmys' pick for Best Actor, his third in a row, and was joined this year by Aron Paul, again terrific as Walter's methhead foil, Jesse Pinkman. Props too to the pin-sharp writing which brought dark laughs and emotional depth to all of the characters.
The hapless Jeese, who could easily have been a two-dimensional facsimile of True Romance's Floyd, grew a heart; Walter just grew a whole new set of problems. Breaking Bad's writer/director, one-time X-Filer Vince Gilligan, kept us guessing with twists and turns he might have learnt from Chris Carter. Can he keep it up? We'll find out when season 4 lands next year.
Nothing short of an addiction in the office, each of Sherlock's precious three episodes was meticulously torn apart by everyone at Empire Towers every Monday morning. You know, in a good way.
Sure, the plots may have bordered on the utterly ludicrous at times (Why did the police keep not noticing people dying in the same cars hired from the same company? Why did the cabbie's victims always pick the wrong pill?), but the characterisation was king, and the public loved it – even topping the Cameron Diaz and Tom Cruise Top Gear special one weekend.
Benedict Cumberbatch's delicate balance of cruel bastardry, high-sprung energy and non-stop logical brilliance never failed to intrigue and entertain, perfectly bouncing off Martin Freeman's Watson – a real, thought-provoking narrator rather than the typically clumsy doctor others have brought to the screen – giving us a modernised mini-series that may have been dragged down by preposterous plotting but still remained must-watch TV.
If anything, the Steven Moffat-ified 21st Century interpretation of the world's greatest detective highlighted its similarities with Britain's other adventuring obsession (and one of Moffat's other success stories), Doctor Who, leading us to wish for some kind of crossover. The Tardis in 221B Baker Street, Holmes and Who chasing robotic bloodthirsty hounds, that kind of thing… Hey, it's an idea!
Let's be honest, on paper, The Trip was a very unlikely success story. A semi-autobiographical account of two comedians going on a jolly around the north of England, eating posh food, getting a bit drunk, winding each other up, and occasionally visiting areas of local interest. Hmm… sounds a bit BBC Four.
And yet, in Steve Coogan and Rob Brydon's hands, it works. Somehow. God knows. The impersonations from the pair of them are the star attraction of course, with their Caines, Winstones and Hopkins(es) sounding ever-so-slightly different from each other, and yet at the same time both remaining… spot-on. Then there's their general inane babbling, rivalling even Seinfeld in the complete comedy bullshit stakes (their waffling on about "I'm going to write a classic, I'm going to write it in an attic" springs to mind) as well as their competitive one-upmanship, culminating in Steve delivering Rob his pre-emptive obituary and refusing to be all that nice. At all.
Directed by Michael Winterbottom and edited into a feature-length sort-of-movie, it even garnered critical plaudits from across the pond at The Toronto Film Festival. Which you can't say about My Family. No sir.
Arguably, the hype surrounding Boardwalk Empire was greater than that of any cable TV show, ever. Scorsese producing, Buscemi starring, it was meant to have cost kersquillions just to advertise (not to mention the price of recreating 1920s Atlantic City in a parking lot), seemingly destined to be the next shining star in the HBO's crown. The good news? It's worth it.
Filling the critical void left by Anthony and the rest of The Sopranos in 2007 was no small task, yet somehow they managed it – in no small part thanks to Buscemi's magnificent master bootlegger, the sneaky, opportunistic, cut-throat Enoch "Nucky" Thompson. Telling tales of alcoholism, decadence, hardship and heartache, 1920s America comes to life in a way that US TV has as yet fail to address – touching on the advent of radio, female emancipation, the pre-depression boom… the list goes on. We almost learnt something, it was bizarre.
But with Sopranos writer Terence Winter at the helm and Scorsese directing the first episode, the show was never going to do anything other than good business – and yet, in the cruellest of ironies, chances are you're going to have to do your own electronic bootlegging to get a look at Nucky and co. any time soon. Our tip? Wait for the DVD. Really, it's that good.
Season 4 of 30 Rock is, in many ways, the same old 30 Rock we know and love. Liz is neurotic and utterly hilarious in equal measure; Jack Donaghy is cynical, slick and much better dressed than you; Kenneth is pagey; Tracy is TRACY, and everything goes on as normal / completely not normal.
Along the way, Tracy tries to win an 'EGOT' (an Emmy award, a Grammy, an Oscar and a Tony award) but finds starring in a Broadway show too much to bear; Michael Sheen makes a cameo that will… well… defy belief (we dare not spoil it for you, needless to say); Al Gore says hi; Jack goes out with two women at once (Elizabeth Banks and Julianne Moore… tough choice, right?) and we get to enjoy all the uproarious antics you'd expect all that to entail.
Essentially, Season 4 of 30 Rock managed to pull off that trickiest of comedy tricks – remaining fresh and new, even with the same characters you know and love. And bar a slightly weak middle, we'd easily put it as our favourite so far. Disagree with us? Well, that's a dealbreaker, ladies! Yes, you just read that. Sorry.
Mad Men is going to Sky for its next season, meaning that the utterly addictive televisual Wednesday one-two of The Apprentice (9pm, BBC Two) then Mad Men (10pm, BBC Four) is, alas, no more.
But, as they say, it was fun while it lasted. Fun and gorgeous and emotional and surprising and magnificent, but fun in that fascinating, almost-historical soapy way. Don's in the city, trying to find out who the real Don is through his work and through the women he meets – and, almost invariably, sleeps with – whereas as Betty is somewhat sidelined, her finest moments coming out of her changing relationship with their daughter Sally, and the therapist they share.
As we head into the mid '60s, it comes as no surprise that the beat era makes itself known, with Peggy making friends with artistic types, and as the newly formed company starts to hit the rocks, stringpurses are tightened and the whole office has to pull together to survive.
A return to form, some have called it, and a great use of a narrative reboot (what with Don leaving home), but more than anything, it's the look of the damn thing. That, and the lack of foot-chopping ride-on lawnmowers this season. Always good, that.
Part Arrested Development, part The Office, part Outnumbered, part real life, Modern Family successfully hoovered up three Emmys earlier this year (beating Glee and 30 Rock in the process), garnered millions of loyal American fans, and since its British premiere earlier this year on Sky, no doubt a few more on these shores too.
As you'd expect from something that's so reminiscent of so many other different shows (and was devised by Frasier writers Christopher Lloyd and Steven Levitan), it's not exactly gamechanging comedy-wise, but it is – and this is the important bit – very funny.
Providing solid belly laughs each and every episode, though it may deal with regular family sitcom tropes it's just so witty you don't much notice. Especially flamboyant and floppy hats off to young Manny (Rico Rodriguez) for his superb scene-stealing skills – and dashing way with the ladies.
"Hamlet on Harleys" would be the easy way of describing Sons Of Anarchy, but in reality, it's far more complicated – and far more brutal – than that handy paraphrasing would lead you believe.
Season 3 deals with the similarly difficult themes as the previous two: fatherhood, loyalty, senile dementia, and, of course, family. There's more than a little Sopranos in here, the balance between bloodshed and political wranglings a delicate one to manage, but stellar lead performances from Ron Perlman, as club President Clay Morrow, and Katey Sagal as his significant other, Gemma (still Leela from Futurama to many) manage to bring the show to an equally addictive and involving level.
In terms of plot, the first few episodes deal with the aftermath of the abduction of the young son of Jax Teller (the club's conflicted pseudo-Hamlet and heir apparent, played by Charlie Hunnam) by a member of the True IRA… but after that, there are just so many twists and turns that anything else would be unfair on you guys.
Rest assured, there'll be just as many heads bashed into just as many slabs of concrete, as well as plenty of emotional moments of tough guy angst as the bikers try to keep their leather-clad dream alive – and provide quality drama while they're at it.
November brought news that Frank Darabont's TV adaptation of Robert Kirkman's beloved zombie comic The Walking Dead was going to get a second series. Hip, and if you don't mind, hooray! Why the middle-class jubilation? Well, because the first series was nothing short of very, very good.
As Stephen King puts it, The Walking Dead was a "21st-century rarity, appointment TV", scoring record-breaking ratings across the world as every man and his undead dog tuned in to get their fill of shambling corpses and see him off Teachers in a big sheriff's hat getting himself into yet another zombie-induced pickle.
If you haven't seen the show yet, and aren't sure whether it's for you, then the first episode should see you right – being as it is arguably one of the best debut episodes of any TV series of late, thrusting you headlong into the post-apocalyptic maws of Cynthiana, Kentucky, and refusing to shy away from the daylight, bringing you wide, cinematic shots of undead-filled disaster zones. The zombie genre hasn't been spoiled with quality TV production like this ever before, so buy yourself the DVD, sit back and enjoy. That's an order.
This was the year that Futurama moved from Fox to Comedy Central, so naturally we were expecting – despite a reduced budget and writing staff – big things. It may have gotten off to something of a stumbling start, but by episode three, Matt Groening's other brilliant animated series began showing familiar signs of greatness with The Attack Of The Killer App. Nicely timed to coincide with the release of the iPhone 4 back in July, it centres on the evil Mom's deployment of her virus-ridden eyePhone.
By week seven, Groening & co. delivered one of the show's best ever episodes. The Late Philip J. Fry has our ginger hero agreeing to test out Professor Farnsworth's time machine right before a big date with Leela. Not only do the pair shoot far further forward than the intended single minute, they learn that they cannot move backwards in time. It's what Futurama does best – big, bold, unquantifiable scientific theory housing some great gags (a bit of Zager & Evans on the soundtrack never hurts, either). And the good news (everyone!) is that 2010 brought us only the first half of season 6; Eps 14 – 26 are slated for 2011. Huzzah!


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Tibet Namtso Lake and Yangpachen Tsurphu Trek at a Glance. Duration 19 nights to 20 days trekking in Tibet. Standard Medium to difficult. Start from Lhasa to Finish Kathmandu. Highest Point Lasar La (5,300m).
Original: http://www.nepaltour.info/tibet-namtso-lake-yangpachen-tsurphu-trek.html
Posted: March 18, 2013, 12:00 am
Nepal is one of the richest countries in the world in terms of biodiversity due to its geographical position and altitude variation. The elevation of the country ranges from 60m above sea level to...


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Press Releases
Aclaris Therapeutics Announces Positive Results in Phase 2 Clinical Trial of A-101 for Treatment of Common Warts
WART-201 was a randomized, double-blind, vehicle-controlled, clinical trial designed to evaluate the safety and dose-response of 2 concentrations of A-101 Topical solution, 40% and 45%, compared with placebo (vehicle) in patients with common warts. Ninety-eight patients were enrolled in the study with 90 patients completing eight weekly treatments at six investigational centers within the United States.
In the trial, the 45% concentration of A-101 demonstrated clinically relevant and statistically significant improvement in the mean change in the Physician's Wart Assessment (PWA) score and in complete clearance of common warts. The side effect profile was similar to placebo.
WART-201 Efficacy Results:
WART-201 Safety Results:
"We are extremely pleased with these results," said Dr.
About Common Warts (Verruca Vulgaris)
Cautionary Note Regarding Forward-Looking Statements
Any statements contained in this press release that do not describe historical facts may constitute forward-looking statements as that term is defined in the Private Securities Litigation Reform Act of 1995. These statements may be identified by words such as "believe", "expect", "may", "plan," "potential," "will," and similar expressions, and are based on Aclaris' current beliefs and expectations. These forward-looking statements include expectations regarding the clinical development of Aclaris' A-101 drug candidate for the treatment of common warts. These statements involve risks and uncertainties that could cause actual results to differ materially from those reflected in such statements. Risks and uncertainties that may cause actual results to differ materially include uncertainties inherent in the conduct of clinical trials, Aclaris' reliance on third parties over which it may not always have full control, and other risks and uncertainties that are described in Aclaris' Annual Report on Form 10-K for the year ended
Contact: Aclaris Contact
Copyright © Aclaris Therapeutics, Inc. All Rights Reserved.


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RUTH WALTON will celebrate her 85th birthday on May 24. In addition to her "sweet shop" days, she was activities director at Horn's Nursing Home and a secretary at Triway Junior High School.
A card shower is planned. Cards and well-wishes may be sent to her at 1926 Lynn Drive, Orrville 44667.


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At this time of year we are drinking more coffee than ever, so this french press was a must. I love that I can make many cups at one time. This Francois et Mimi 50 Ounce Large-Sized Double Wall Insulated French Coffee Press has worked wonders for us, the coffee comes out just so amazing! This has made the taste in the coffee just come alive.I was very impressed with this French Press. I love that it is stainless steel, it makes it so classy looking. I was hooked the very first time of using this. They are easily cleaned, and they do not keep your finger smudges. These are made from a higher quality, They are extremely nice and well made. Also, these are very durable. I received these at a discounted price for my honest opinion.
Get your very own here: http://www.amazon.com/dp/B00UM589IS
Monday, April 6, 2015
REVIEW- Francois et Mimi 50-Ounce Large-Sized Double Wall Insulated French Coffee Press
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Hey there, time traveller!
This article was published 12/1/2013 (1683 days ago), so information in it may no longer be current.
THE Winnipeg Jets will try to get things started with a bang today at the MTS Centre, inviting the public to watch their first official post-lockout practice.
The team will work out between noon and 1 p.m. Admission is free, though subject to the arena's seating capacity of 15,004, and doors will open at 11:30 a.m.
Today, every fan in attendance will receive a voucher for one complementary 24 oz. fountain beverage and one complementary regular popcorn.
The team's official merchandise store, Jets Gear, will be open and offering all merchandise at a discount of 25 per cent off regular prices today.
The Jets' official training camp will be a short one, beginning today and lasting until Friday.
Twenty-four players are expected to be on the ice today.
The Jets are listing defencemen Zach Bogosian and Derek Meech on their training camp roster but both players are sidelined with injuries.
After today's action, the team will practise each morning the rest of the week at the MTS Centre, with the exception of Wednesday at the MTS Iceplex.
The skates at the MTS Centre are not scheduled to be open to fans. Wednesday's workout at the MTS Iceplex at 10:30 a.m. will be available for public viewing.
-- Tim Campbell


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11 oz. C-Handle Mug - White
Rated 4.5 /5 based on (9)
Blue, Navy
Blue, Royal
Green, Dark
Green, Forest
Green, Hunter
Green, Kelly
9 customer reviews
5/5 stars
Published 8/15/2013 by Janet John (Clinton Township, Michigan)
I needed a promotional item FAST! InkHead support was phenomenal! They approved and put a free rush on the order and shipped it quickly without outrageous shipping charges. The product was received in plenty of time to prep it for a planned event.
A detailed logo was printed on both sides of the mug with no extra charge--I loved that! There are a disclaimer saying that the details might not appear correctly. This was far from the truth! All of the logo details were there and the navy color I ordered was perfect! I am so happy with these mugs and will order them again in the future.
2 out of 2 people found the following review helpful. Was this review helpful to you?
4/5 stars
Published 6/26/2013 by Barb (Ft Lauderdale, Fl)
Our customers love these, which means that WE love them too! They are inexpensive and sturdy, which is always a plus! And they are VERY quick to delivery. This is a great company with good products, good prices and fast/efficient service. That's the hat trick of "it works" for us!!
1 out of 1 people found the following review helpful. Was this review helpful to you?
Published 6/20/2017 by Joe P. (La Mesa, CA)
These mugs are awesome! We use them for in-store drip coffee and for gifts. They are super durable and built to last. I recommend them to anyone thinking about it.
0 out of 0 people found the following review helpful. Was this review helpful to you?
Published 10/20/2014 by Jordan (New London, CT)
We love our customized mugs! The two colors from our design showed up quite well on the white mugs. The customer service was phenomenal - the representatives worked with me on very short notice to make sure my final product got to me in less than two weeks (and with free shipping, too!). Can't recommend the customer service enough, and I will definitely recommend Inkhead to all my friends!
Published 10/02/2014 by Robin (New York, NY)
These mugs are great! They are good size, strong weight, and have a good sized handle. And for the price, we absolutely can't think of using anything else!
Published 3/07/2014 by Katie (Berkeley, CA)
The mugs look great. Clean, simple, and high quality. Perfect!
Published 7/26/2013 by Fallstop (Montreal, QC)
Great price and does what it's suppose to do. Clean look.
Published 1/22/2013 by Dom (Houston, TX)
The mugs have a good size, the printing came out well. The packaging is very good so that all mugs made it without any problem.
Overall very happy with it.
Published 5/24/2011 by RD (Austin, Texas)
Simple, useful, and inexpensive. A perfect way to keep your company info floating around a customer's home or office for years to come.
0 out of 1 people found the following review helpful. Was this review helpful to you?
9 reviews


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One of our favorite things to do when the kids are off track is to have fun breakfasts together.
When school is in session it is hard for us to find the time (or the bonus energy) in the morning to make a fancy breakfast. But when our mornings can be stretched out longer than an hour we LOVE working together to create a pseudo-gourmet feast in the morning.
One of our favorite morning meals is Monkey Bread.
We have bought it frozen several times, but for Christmas I got this awesome cookbook that had a recipe to make it for ourselves. I had to make a few adjustments to accommodate the size of our family and our personal preferences, but it is super delicious and not hard to throw together at all.
Here's how it's done:
1 1/2 cup sugar
2 Tbsp cinnamon
4 (7.5 - 8 oz) cans of refrigerator biscuits
1/2 cup butter
1/2 cup brown sugar
1 Tbsp honey
(This recipe easily serves all eight of us.)
1. Preheat your oven to 400 degrees F. Spray your friendly neighborhood bundt pan with cooking spray and set it aside.
2. Mix the sugar and cinnamon into a gallon sized zip seal bag and shake it a bit to mix it.
3. Open your refrigerator biscuits (and if you are like me cringe while you hold the paper opening strip and shake them as far from yourself as possible to get them to open because you HATE that pop more than almost anything else in the world) and cut them into fourths with kitchen scissors.
4. Add these dough blobs a few at a time to the zip seal bag and shake them to coat them with the sugar/cinnamon mixture (seriously, only add a few at a time because if you just dump them all in there they moosh together and don't get coated and they turn into one gigantic dough ball).
5. Place your coated dough into your bundt pan. Don't just dump them from the bag because you will likely have extra sugar and you don't want it all over in your pan.
6. In a separate microwavable bowl add your butter, brown sugar and honey. Put these in the microwave for about 1 minute and stir together (they should be melted when you are done).
7. Slowly drizzle your butter/sugar syrup over the top of the dough in your bundt pan. Be sure to get some syrup on every part of the bread.
8. Bake for 20 minutes or until he brad is golden brown. Take it out of the oven and let it cool for a few minutes (the syrup coating will be REALLY hot and will burn your mouth...I promise, I've learned from experience). Invert your pan and let your delicious concoction fall out onto a plate (it comes out really easily).
Now for the best part...
You can't beat a table full of adorable kidlets happily eating a tasty breakfast together. Gotta love life as a family!


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found a new home!
Plenty of my friends are looking
for one too. Find a pet to adopt.
I am already neutered, housetrained, up to date with shots, good with kids, and good with dogs.
If you are interested in any of our animals please take the time to go to our website and fill out an application. Follow this link to our application: http://www.heavencanwaitanimalrescue.com/adoption-application.html
*Adoption Fee*
$200.00 includes the following: neuter, parasite free, Rabies vaccination, DHLPP , vaccination, Bordetella vaccination, microchip, heartworm negative and current on prevention.
Thanks for choosing adoption!!!!
Petie, a Russellville Pit Bull Terrier dog was adopted! Here's how you can benefit by looking at adoption as an option to buying a Pit Bull Terrier for sale in Russellville, or seeking a Pit Bull Terrier dog from a breeder in Russellville.


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Dress - eBay (originally ASOS Curve), Leggings - Newlook Inspire, Socks - ASDA, Boots - Newlook (Wide Fit), Necklace - Charity Shop, Lipstick - MAC, Nail Varnish - Barry M, Ring - My Grandfather's, Scrunchie - Urban Outfitters.
I found this dress on eBay and I absolutely adore it! The fit is so flattering and comfortable, the collar and cuff detailing is perfect and the print is fabulous. I really cannot recommend it highly enough. The nipped in, elasticated waist adds the perfect shape to the dress and the pleating on the bust allows ladies with a larger bust, like myself, to wear the dress without worry of the material pulling.
I love these boots! I don't wear heels usually because I'm 5'7 barefoot so I feel a little uncomfortable in them, but these are just the right height. I don't need to purchase from the wide fit section on the whole but I adored these boots when I saw them so I bought them anyway and the extra room makes them amazingly comfortable! I'm a sucker for the gold panel detailing around the heel too. My little socks were £1.50 from ASDA! You can't see the cute gold flecking through them in this photo but they are great to wear visibly with boots/shoes!
Now it know these reds are not entirely coordinated but I love them both! This Barry M red glitter nail varnish is amazing, especially when used over a solid red. It last forever and is totally chop resistant! For a while I thought I'd lost my MAC lipstick and I was very sad because this is THE perfect shade, it's Lady Danger and I love how matte it is! It lasts all night and is even drink proof.
Finally, here is a photo of my man and I on the train before Billy Bragg, cheering it up haha!
Lots Of Love,


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Chase Club
An exclusive ambiance in a relaxed and classically-styled space
Locals know where to come when they're craving gastropub cuisine with gourmet flair and a stellar "Liquid Art" cocktail. And now you do, too. Overlooking our Mediterranean-style pool and terrace, Chase Club is a Central West End favorite. Lunchtime artisan burgers are second to none and late-night appetizers and live music on weekends are perfectly paired. Plush sofas overlook the beautiful pool and an outdoor fireplace sets a cozy tone on cool evenings. Relaxed yet sophisticated, Chase Club is the perfect place for lunch, dinner, a nightcap, or soaking up some local culture.
Sunday: 11AM - Midnight
Monday - Saturday: 11 AM - 1:30 AM
Chase Club Food Menu
Chase Club Drink Menu
Chase Club Late Night Menu


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Scene Pictures
Join Pride Studios here today for just $1!
Javier and Aiden have been dating for a few weeks and they are both really into each other. Javier invites Aiden over for the first time since they've been seeing each other to take their relationship to the next step in Putting Out at Pride Studios. The cameras are rolling as they explore each other's bodies and get into a 69 suck session before the foreplay leads to Javier fucking Aiden's hole!


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Release Your Hip Flexors: Groin Stretch 101
The kneeling groin stretch, also known as the Sampson stretch, is a great way to stretch the hip flexors. You might think you know this stretch, but with a little fine-tuning, you can take this one to a whole new level. Just follow these steps and feel the difference for yourself.
Hip Flexor Release
- Kneel on your left knee with your right foot out in front. If kneeling is painful, then place some extra padding under your knee.
- Make sure your right heel is out in front of your right knee and your back foot is in line with your thigh.
- Now, we are going to square up your hips. Place your index fingers on your ASIS, the bony prominences at the front of your pelvis. (By the way, ASIS stands for anterior superior iliac spine, but that's kind of a mouthful.)
- Make sure that your left and right ASIS point straight ahead, like two headlights on a car. We are trying to eliminate any rotation in your pelvis.
- Press the big-toe joint of the back foot into the ground. This will have a neurological impact on the psoas because it is in its most elongated position and pushing the big toe joint into the ground activates an isometric contraction at length.
- Push the back knee and the heel of the front foot into the ground. This activates the lateral hip stabilizers, which help the hip stabilize its position to help the psoas do its thing.
- Be sure to have your pelvis in a neutral or posterior position, but not in an anterior position.
At this point you will feel work in your hips and a stretch in your hip flexor. Hold for one minute, then switch sides and repeat.
Find out more about hip health:
Topic: Fitness
See more about: groin, hip flexors, hip stretch, hip mobility, muscle soreness, yoga, mobility, videos, videos


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Note: All prices in US Dollars
Why purchase from us?
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Marble Pet Plaques (oval)
These quality black marble plaques make beautiful memorials for your beloved pet that can be displayed on a desk, or in a bookcase as a tribute to your best friend that will last forever. Your pet's photo and 1 or 2 lines of custom text are permanently laser engraved onto the surface using the latest computer technology. A stand is included with each plaque.
If you would like parts of the picture removed from the background (ie. tree, house, person etc.) just tell us what you want removed in the e-mail when you send your photograph.
5" x 7" w/stand $97.50
8.5" x 11" w/stand $119.50
Photo Submission:
You will receive an order number when you place your order.
You may e-mail your digital photo, preferably in jpeg or gif format. Other formats may take more time to convert for best quality imaging.
Send the digital image to us at firstname.lastname@example.com and reference your order number in the e-mail.
If you are sending a hardcopy photograph send only original photographs. Do not send prints or photocopies of photographs, or prints made from a laser or ink-jet printer. We need the highest quality image to work with to provide you the very best reproduction possible to memorialize your beloved pet and family friend.
The original should be mailed to us, with your order number referenced at:
RMH Online
14260 W. Newberry Rd. #218
Newberry , FL 32669-2765
Your Original will be returned to you.
Please allow 7 to 10 days for production


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Bodog leaving US market at end of year
Chalk up yet another US online poker site to the after effects of Black Friday. On Tuesday Bodog announced it would allow its gaming license with Morris Mohawk Gaming to expire at the end of the year, signaling the end of US operations.
Long known for thumbing their nose at US gaming laws, Bodog recently received a UK gaming license and will now likely be focusing their attention on the European market, and coupled with the litigious climate in the US market, Bodog has now joined a growing list of online poker providers who have said thanks but no thanks to the largest market in the industry, the US.
Here is the statement that was released on CalvinAyre.com explaining the decision to leave the US online poker market:
"Following recent news that Bodog UK has been granted a gambling license by the UK Gambling Commission, we are pleased to announce further developments to the Bodog brand going forward.
As highlighted last week, Bodog UK's CEO, Patrik Selin believes the trust and credibility that having a UK license brings to the brand will benefit customers and allow him to attract the best talent in the industry to work with him.
However, in order to ensure the brand's expansion is not affected by negative perceptions, both in the UK and elsewhere in emerging markets such as Asia, where Bodog88 is already successful, a decision has been made to withdraw the Bodog brand from the US market at the end of 2011. As a result, the Morris Mohawk Gaming Group's (MMGG) brand licensing agreement will be allowed to expire on 31 December, following which US residents will no longer be able to access bodog.eu or any other Bodog branded website. We understand that MMGG will launch under its own brand in 2012, licensed by the Kahnawake Gaming Commission. All Bodog.eu clients' funds will remain safe and they will have the option to switch to a new MMGG brand should they wish but MMGG confirms it's business as usual.
This decision presents a fresh direction for the Bodogbrand.com as it looks to create a new chapter in its history. The move will enable the Bodog brand to continue its growth and expansion and maintain its position as the world's largest gaming brand."
Tags: Black Friday, bodog, Chalk, Credibility, Emerging Markets, Gambling Commission, Gaming Group, Gaming Laws, Gaming License, Kahnawake Gaming Commission, Licensing Agreement, Mmgg, Mohawk, Negative Perceptions, Online Poker, Patrik, Selin, Thanks But No Thanks, Uk Gambling, Uk Gaming
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Average Rating: 4.5/5 stars (3 reviews)
100% would recommend it to a friend.
This Item is no longer available at Scrapbook.com.Write a Review
I love this little stamp! And such a good deal for the price.
I make a lot of cards for Operation Write Home, and this little stamp is perfect for that. I used this stamp on a note card for our military troops. I think it turned out cute!
You can see that card in the gallery along with a few other cards that utilize the stamps I've gotten at Scrapbook.com!
I actually bought this set for the little bird but I am really loving the larger bird. It looks great with a little added color. Very clear and detailed stamp.
Used in this layout: Happy Boy
Needed a cute bird stamp and found it here! Minimum coloring required, but still cute.


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text: English-Style Pale Mild Ales are light amber to medium amber. Chill haze is allowable at cold temperatures. Fruity-ester aroma is very low to medium low. Hop aroma is very low or low. Malt flavor dominates the flavor profile. Hop flavor is very low to low. Hop bitterness is very low to low. Very low diacetyl flavors may be appropriate in this low-alcohol beer. Fruity-ester flavor is very low to medium low. Body is low to low-medium.


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Obesity, by definition, is the extreme excess of body fat. Over the past two decades, obesity has become an epidemic that can only be prevented through proper diet and exercise. Because of the huge advancement in technology, the American lifestyle has become more and more sedentary, leading to the obesity problem. Now, we even see children below the age of 13 become epidemic; we would not have seen this 30 years ago.
By 2010, obesity rates double from the 1980s to 20%, with some states as high as 30% obesity rate. A lot of this can be blamed on the lack of movement in a person's life and the highly processed/unnatural food that is consumed. Although the American diet has cut sugar usage down by 28%, we have skyrocketed corn based sweetener usage to 500% of what it used to be.
This has affected our culture through a more conscientious awareness of what we are put into our bodies. Recently, Michelle Obama has had legislature passed as to what defines a school lunch and what each school lunch should provide.
Social Media
Over the past decade, we have seen the introduction of huge amounts of social media. Twitter, Facebook, Instagram, Vine, and many more have begun to consume the life of users that log on. Most social media has been wired and specialized for the use on mobile devices and with the increasingly mobile world, that makes social media all the more appealing and entertaining.
Google is the most used search engine the world has ever seen. Most likely, you can't go a day without Googling something because the database behind Google has such a huge amount of resources to answer a question. Created at Stanford University in 1996, Google was the first and only search engine to organize results by relevance to the question. Google is still the premier search engine.


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Language Direction Problem (RTL – LTR)
First i want to say thank you for this great plugin.
but unfortunatly, i have a language direction problem with polylang (v0.9.4).
I am using it on a multisite wordpress. i created a site with 2 languages. Persian (which is RTL) & English (LTR).
i don't have any 3rd plugins in multisite and there are only default wordpress plugins + polylang.
also i am using TwentyEleven Theme.
Persian is my deafult Language and when the site loads, in html tag i see the correct values in "dir" & "lang" properties. ( <html dir="rtl" lang="fa-IR"> )
but when i switch to English, "dir" property value is not being changed and it's still on "rtl", but the lang propery is correct (en-US). therefore the correct css styles are not being loaded.
settings in polylang admin panel are correct for each language. (english is set to left to right & persian is set to right to left)
i am using default permalinks.
i unchecked to set language base on browser prefrenace.
"The language is set from content. Posts, pages, categories and tags urls are not modified." is choosed.
i aslso unchecked "Hide URL language information for default language". (checking this hadn't fixed the problem)
I think there are some problems in polylang core.php on lines 325 – 328.
beacause when i manually add the following code after last else on this section (line 346 between two "}") , the problem is fixed:
$my_new_value = new Polylang_Core(); $my_new_value_lang=$mori->get_current_language()->description; if($my_new_value_lang == 'en_US') $GLOBALS['wp_locale']->text_direction = 'ltr'; else $GLOBALS['wp_locale']->text_direction = 'rtl';
so it will look like this:
// reinitializes wp_locale for weekdays and months, as well as for text direction unset($GLOBALS['wp_locale']); $GLOBALS['wp_locale'] = new WP_Locale(); $GLOBALS['wp_locale']->text_direction = get_metadata('term', $this->curlang->term_id, '_rtl', true) ? 'rtl' : 'ltr'; // translate labels of post types and taxonomies foreach ($GLOBALS['wp_taxonomies'] as $tax) $this->translate_labels($tax); foreach ($GLOBALS['wp_post_types'] as $pt) $this->translate_labels($pt); } // and finally load user defined strings $GLOBALS['l10n']['pll_string'] = $this->mo_import($this->curlang); } } else { // can't work so load the text domains with WordPress default language foreach ($this->list_textdomains as $textdomain) load_textdomain($textdomain['domain'], $textdomain['mo']); } $my_new_value = new Polylang_Core(); $my_new_value_lang=$mori->get_current_language()->description; if($my_new_value_lang == 'en_US') $GLOBALS['wp_locale']->text_direction = 'ltr'; else $GLOBALS['wp_locale']->text_direction = 'rtl'; }
but this is an ugly & temporary fix.
i don't know if it is a bug or i am doing something wrong.
so i need your help.
thank you.
- The topic 'Language Direction Problem (RTL – LTR)' is closed to new replies.


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Orange + Basil Lip Balm
Shimmering Skin Perfector Opal Glow on the Go Collection
Backlight Priming Filter
Illumify Shimmering Highlight
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WHAT IT IS A powder shadow designed for quick and easy application.
BENEFITS FOR YOU • Longer-lasting wear. The powder formula was made to wear all day long. • Pigment packed. The pigment rich formula delivers bold color, yet is light and buildable. • Non-drying formula. These shades are easy to wear, buildable and won't dry out the eye lid. • Pressed formula. Avoids heat exposure and preserves the integrity of the raw ingredients. • Organic and vegan.
YOU'LL EXPERIENCE This non-drying formula sweeps across your eyes, delivering a color pop that will soothe and beautify your eyelids.
WHY IT'S GLOWING This isn't your typical eye shadow. This formulation was created with you in mind. Unlike other formulas, this pigment-packed shadow is buildable, long-lasting and it doesn't dry out your eyelids! That's seriously amazing. RMS Beauty delivers a product that not only looks good, but does good too - it's organic and vegan!
WHAT'S NOT IN IT Formulated without parabens, sulfates, phthalates and gluten.


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Dusk Treasure Escape
TheEscapeGames:-Bonny Modern Room Escape is an another point and click type excitement house escape game developed by theescapegames.Imagine,one person was trapped inside the bonny modern room.He don't know, how to escape from this room.He was panic.So,using your fantasy skills to rescue the person.You can easily rescue the person from trapped modern room.The Escape games have more exciting
and amusing free online escape games.Have more fun.Good Luck. The motivation of this game has to escape from the trapped bonny modern room.Use your smart skills.Have more fun.good Luck.
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