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Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease (CLD) and hepatocellular carcinoma (HCC). HBV encoded X antigen, HBx, and pathways implicated in the self-renewal of stem cells contribute to HCC, but it is not clear whether HBx expression promotes "stemness." Thus, experiments were designed to test the hypothesis that HBx triggers malignant transformation by promoting properties that are characteristic of cancer stem cells (CSCs). To test this hypothesis, HepG2 cells were stably transduced with HBx and then assayed for phenotypic and molecular characteristics of "stemness." The relationship between HBx and "stemness"-associated markers was also evaluated by immunohistochemical staining of liver and tumor tissue sections from HBV infected patients. The results showed that Oct-4, Nanog, Klf4, β-catenin and EpCAM were activated by HBx in vitro and in vivo. EpCAM was detected in the nuclei of human HCC cells from infected patients. HBx promotes "stemness" by activating β-catenin and epigenetic up-regulation of miR-181, both of which target EpCAM. HBx expression was also associated with depressed levels of E-cadherin. Moreover, HBx stimulated cell migration, growth in soft agar, and spheroid formation. This work is the first to propose that HBV promotes "stemness" in the pathogenesis of HCC. HBx associated up-regulated expression of multiple "stemness" markers support the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristics of CSCs.
HBV associated HCC is among the top five most frequent cancers worldwide (1). About 250,000 new cases of HCC are diagnosed yearly, and most people die within a year of diagnosis. The mechanism of HBV mediated HCC is not clear. However, HBV DNA is integrated, often highly rearranged, within host DNA in tumors and tumor-derived cell lines (2). These templates frequently produce HBx (3) that is active in trans-activation assays (4). Sustained production of HBx is associated with hepatocellular transformation, and represents a major contribution of HBV to HCC (5). HBx RNA and protein are highly expressed in chronically infected livers and strongly correlate with CLD (6). HBx has transcriptional trans-regulatory properties that alter patterns of host gene expression (2, 6), inhibits proteasomal degradation of growth regulatory proteins (7), stimulates cellular kinases that alter signal transduction (5), and utilizes epigenetic machinery (8). This suggests that HBx mediated tumor development involves epigenetic mechanisms that become operative during CLD.
Microarray analysis of human HCC specimens, cancer cell lines, and transgenic models established the molecular similarities between CSCs and hepatic stem cells (HSCs), and highlighted the importance of CSCs in the prognosis of liver cancer (9). "Stemness" transcription factors (e.g., Oct-4, Klf-4 and Nanog) re-expressed in cancer cells (10), and their reactivation contributes to tumorigenesis in somatic tissues (11). Epithelial cell adhesion molecule (EpCAM) is a marker of HSCs and CSCs (12) and acts as a mitogenic signal transducer via proteolysis and nuclear translocation of its intracellular domain, EpICD. EpICD binds to DNA in a complex with the scaffolding protein FHL2, β-catenin, and Lef-1, and regulates cell proliferation (13). EpCAM sustains "stemness" through EpICD binding to c-myc, Oct-4, Nanog, and Klf-4 promoters (14). The EpCAM promoter has Tcf-binding sites and is a transcriptional target of β-catenin (15). EpCAM was also regulated by miR-181. Forced expression of miR-181 endowed EpCAM-positive HCC cells with stem cell properties, whereas miR-181 suppression induced differentiation (16).β-catenin, like EpCAM, is important in stem cell self-renewal and in carcinogenesis (17), in part, by binding the promoters of Oct-4 and Nanog (18). Aberrant activation of their self-renewal program by β-catenin may contribute to the appearance of CSCs (19). Importantly, HBx also transcriptionally activates β-catenin in up to 80% of HCCs (20). This underscores the potentially close relationship between HBx, activated β-catenin, and stem cell renewal.
β-catenin acts as a transcriptional activator in the nucleus, but promotes cell adhesion when bound to E-cadherin (21). E-cadherin provides a physical link between adjacent cells and is crucial for cell polarity and the structural integrity of tissue (22). Altered expression and cellular distribution of E-cadherin is frequently associated with invasiveness in human cancers including HCC (23). In this context, HBx suppresses E-cadherin expression by promoting methylation of the E-cadherin promoter (24). This event results in the stabilization of β-catenin through tyrosine phosphorylation mediated by src, which is also activated by HBx (25). Collectively, these findings suggest that HBx promotes the development of HCC by triggering the expression of "stemness" factors in the chronically infected liver. The results of the present study support this hypothesis.
The human hepatoblastoma cell line, HepG2, was stably transfected with HBx (HepG2X) or the bacterial chloramphenicol acetyltransferase (control) gene (HepG2CAT) as previously described (26). Cells were cultured in MEM (Invitrogen, Carlsbad, CA) supplemented with 100 mM nonessential amino acids, 100 mM sodium pyruvate, and 10% fetal bovine serum (Invitrogen), at 37°C in a humidified 5% CO2 incubator.
Forty-three formalin fixed, paraffin embedded paired tumor (HCC)/nontumor (adjacent liver) tissues were obtained from Chinese patients who underwent surgery at the Third Military Medical University, Chongqing, China. All patients were hepatitis B surface antigen (HBsAg) positive in blood; 41 were males, the age range was from 35–69 (average: 48), and all were of Chinese ethnicity (Suppl. Table 1). Additional snap frozen tumor/nontumor pairs from 20 HBV infected patients were obtained from Queen Mary College at the University, Hong Kong, China. All patients were also positive for HBsAg in blood; 18 were male, and their ages ranged from 33–71 years (average: 50.5) (Suppl. Table 2). All samples were used for diagnostic purposes, and then used for this study. Ten uninfected human liver tissues (Abcam, Cambridge, MA) were used as controls. The use of samples was approved by the Institutional Review Boards at all participating universities.
Single cell suspensions of HepG2X and HepG2CAT cells were plated at a density of 1 × 103 cells in 2 ml of medium in 6-well Ultra-Low Attachment Microplates (Corning, Corning, NY) and maintained for up to 16 days. Spheroids were observed and counted using an ECLIPSE Ti inverted microscope operated with a Nikon DS-Fi1 camera and NIS Elements software (Nikon, Melville, NY).
Single cell suspensions of 1 × 104 cells were mixed with 0.3% agar (Sigma, St. Louis, MO) in complete growth medium and seeded in triplicates into 6-well plates coated with 0.5% hardened agar. Plates were incubated at 37°C for 28 days. Colonies ≥1 mm in diameter were counted under code by light microscopy.
Single cell suspensions of 1.5 × 105 cells were plated in triplicates into 6-well BD BioCoat™ Matrigel™ Invasion Chambers (BD, Franklin Lakes, NJ) according to enclosed instructions. Cell migration was observed after 24h by hematoxylin and eosin (H & E) staining.
For tumorigenicity studies, two groups of 10 six-week-old nude mice (Charles River Laboratories, Wilmington, MA) were injected subcutaneously at a single site with 6 × 106 HepG2X or HepG2CAT cells. Tumor onset was scored visually and by palpitation independently by two trained lab personnel. Tumor sizes were determined by wet weight at the time of euthanasia (6 weeks). These experiments were approved by the Institutional Animal Care and Use Committee at Temple University.
Cells were lysed in Cell Lysis Buffer with protease inhibitor cocktail (Cell Signaling, Danvers, MA). Nuclear extracts were prepared using Nuclear Extract Kit (Active Motif, Carlsbad, CA) according to enclosed instructions. Protein extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Sanford, ME). The membranes were incubated overnight in 5% nonfat milk in Tris Buffered Saline/0.1% Tween-20 with primary antibodies against HBx, Oct-4, E-cadherin, β-catenin, Lamin A and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), Nanog (Cell Signaling, Danvers, MA), Klf-4 and EpCAM (Abcam), Ac-Lys H3 (Active Motif). The blots were developed using the ECL plus detection kit (Amersham, Piscataway, NJ) and exposed to Kodak imaging films (Kodak BioMax, Rochester, NY). Images were processed using ImageJ software (NIH).
Suspensions of 3.5 × 105 cells were plated in 35-mm culture dishes with or without 350 nM TSA (Sigma) in 2 ml of medium. Extracts were collected at 12 and 24 hours and analyzed by western blotting.
PowerBlot analysis of HepG2X and HepG2CAT cell lysates was performed by BD Transduction Labs (Franklin Lakes, NJ). Briefly, 400 μg of cell lysates were analyzed by SDS/PAGE, and transferred to Immobilon P (Millipore, Billerica, MA). The membranes were blocked with 5% nonfat milk. Each membrane was cut into vertical strips, and each strip was incubated with a complex antibody cocktail. Signals were developed by chemiluminesence and measured using PDQuest 2-D analysis software (Bio-Rad, Hercules, CA). For each of 3 experiments, 3 gels were run using HepG2X and HepG2CAT cell lysates.
Small RNAs were isolated from HepG2X and HepG2CAT cells and 20 pairs of HCC tissue samples using Ambion (Austin, TX) mirVana miRNA isolation kit according to enclosed instructions. Microarray analysis was performed with miRNAs isolated from HepG2X and HepG2CAT cells by LC Sciences (Houston, TX). Differentially expressed miRNAs in cells were identified by Cy3(HepG2X)/Cy5(HepG2CAT) ratio, and those with a p-value < 0.01 were considered for further characterization. Differential expression of selected miRNAs in cells and tissues was validated by qRT-PCR using a Smart Cycler System (Cepheid, Sunnyvale, CA). The ΔΔCt calculation was done as follows: ΔΔCt = ΔCt of miR-181a in tumor–ΔCt of miR-181a in non-tumor tissue. Briefly, the samples with 25 ng small RNA were first denatured at 95 °C for 3 min and then run for 40 cycles (95 °C for 15 sec and 60 °C for 30 sec). U6 was used for normalization.
Tissue sections were deparaffinized, dehydrated, treated with Uni-TRIEVE antigen retrieval (Innovex, Richmond, CA) and stained using the UltraVision Detection System (Thermo Scientific, Rockford, IL) according to enclosed instructions. Antibodies used for staining were the same as those described above for western blotting except for anti-HBx (anti-99 custom made antibody) (27) and anti-EpCAM (Millipore, Billerica MA). Normal mouse or rabbit IgG (Vector Labs, Burlingame, CA) were used to rule out false-positive responses. Pre-absorption of primary antibodies with corresponding antigens was performed on tissue sections to insure specificity.
Statistical values for cell migration, growth in soft agar, and tumorigenesis were defined using an unpaired Student's t-test, in which P <0.05 was considered significant. The relationship between HBx and EpCAM, β-catenin, and E-cadherin obtained by immunohistochemistry was determined using 2 × 2 comparisons in the Chi square (χ2) test. Statistical significance was considered when P < 0.05.
PowerBlot analysis was conducted using whole cell lysates from HepG2X and HepG2CAT cultures. The results showed 4.5 ± 0.5-fold up-regulated expression of β-catenin in HepG2X compared to control cells (Fig. 1A–B). Several β-catenin target genes were also up-regulated, including MDR1 (7.8 ± 1.8-fold; Fig. 1C–D) and c-myc (4.7 ± 0.7-fold; Fig. 1A–B). HepG2X cells expressed 6.7 ± 1.2-fold lower levels of E-cadherin (Fig. 1C–D) compared to HepG2CAT cells. Selected markers were then further characterized by western blotting (Fig. 1E–F). In whole cell extracts, HBx was associated with down-regulation of E-cadherin (3.8-fold), as well as up-regulation of β-catenin (2.5-fold) and EpCAM (3-fold). EpCAM was also up-regulated in HBx-expressing Hep3B and Huh7 cells (Suppl. Fig.1). In HepG2 cells, β-catenin is present as wild type (upper band) and truncated mutant (lower band) (Fig. 1E) due to partial exon 3 deletion (28). HBx up-regulated wild type β-catenin without modifying the levels of truncated β-catenin (Fig. 1E). Previous work confirmed that nuclear β-catenin in HBx positive HCC is wild type by DNA sequence analysis of β-catenin exons obtained from paraffin blocks (29). HBx-associated up-regulation of β-catenin has been demonstrated in Huh7 (30, 31) and Hep3B cells (20, 31). Down-regulation of E-cadherin in Hep3B cells has also been demonstrated previously (24). Moreover, HBx-mediated repression of E-cadherin was associated with activation of β-catenin in Hep3B cells (24). In nuclear extracts, HBx was associated with up-regulation of Oct-4 (1.6-fold), Nanog (3.4-fold), and Klf-4 (3.5-fold) (Fig. 1F). These data suggest that HBx is associated with the re-activation of "stemness" transcription factors Oct-4, Nanog, and Klf-4, and "stemness" associated EpCAM and β-catenin.
Additional experiments were conducted to determine whether the results of PowerBlot analyses and western blotting could be validated in vivo. Thus, clinical samples containing HCC and nontumor liver were stained for HBx, Oct-4, Nanog, β-catenin, EpCAM and E-cadherin (Suppl. Table 1). Among 43 patients who underwent surgical resection for HBV associated HCC, 31 had both tumor and adjacent nontumor liver, 9 had only tumor in their blocks, and 3 patients had only nontumor liver. Among these, HBx staining was observed in 26 of 40 tumors (65%) and in all 34 nontumor livers (100%). In 82% of cases with tumor and nontumor tissues, HBx staining was stronger and more widespread in liver compared to tumor, as previously reported (32). Ten commercially available liver sections from uninfected individuals were uniformly negative for HBx. HBx staining was cytoplasmic in all cases (Suppl. Fig. 2), and one case showed nuclear staining in nontumor liver (data not shown). EpCAM was present in 26 cases in the tumor compartment (66%). Membranous EpCAM staining (Fig. 2A) was observed in the nontumor liver among 21 patients (60%) (Fig. 2C), even though EpCAM is not expressed in uninfected, healthy liver (12). In tumors from 3 patients, nuclear EpCAM was also detected (Fig. 2B). With regard to β-catenin, staining was observed in the tumor compartment from 28 patients (74%), and in the nontumor compartment from 19 patients (61%). Although membranous β-catenin is characteristic of normal, uninfected liver, all cases of tumor and nontumor tissues showed both membranous and cytoplasmic staining for β-catenin, consistent with β-catenin activation, as previously shown (29). β-catenin was also observed in the nuclei of two tumors (data not shown). In contrast, E-cadherin staining, which was exclusively membranous, was detected in only 7 tumors (18%), but was present in 20 cases where nontumor liver was available (74%). These results are similar to those previously published (24). When staining was performed for Nanog and Oct-4, six and two tumor samples were positive in scattered nuclei, respectively (Fig. 2D and E). Hence, "stemness" and "stemness"-like markers were observed in HBV associated HCC.
Additional analyses were conducted to determine the relationship between HBx and these markers. In the tumor compartment, co-staining between HBx and EpCAM was seen in only 16 cases (Fig. 3A–B) (χ2 = 2.03; P > 0.1), between HBx and β-catenin in 19 cases (χ2 = 0.23; P > 0.5), and between HBx and E-cadherin in 6 cases (χ2 = 0.88; P > 0.3). Hence, there appears to be no correlation between HBx and these markers in tumors. In contrast, in the nontumor compartment, where HBx is strongly expressed, co-staining between HBx and EpCAM was seen in 21 cases (χ2 = 11.8; P < 0.001), between HBx and β-catenin in 19 cases (χ2 = 5.88; P < 0.02), while an inverse correlation was seen between HBx and E-cadherin in 20 cases (χ2 = 6.04; P < 0.02), suggesting a strong correlation between HBx and these markers. Other blocks of chronically infected liver from patients without HCC were also evaluated, but the results obtained were the same as in adjacent liver tissues from HCC patients (data not shown).
Analysis of relationship between β-catenin and EpCAM staining and HCC grade showed an inverse correlation between EpCAM staining and HCC in grade 1 (χ2 = 10.2257; P < 0.005) and grade 2 (χ2 = 5.3831; P < 0.025), but not in grades 3 (χ2 = 1.3; P > 0.05) and 4 (χ2 = 1.06; P > 0.3). There was no correlation between β-catenin staining and tumor grade. Experiments with a larger number of samples should be conducted to address these relationships more thoroughly.
MicroRNA(s) are known to contribute importantly to the pathogenesis of many tumor types (33). When miRNA microarray analysis was performed on HepG2X and HepG2CAT cells, HBx was associated with up-regulation of miR-181a (2-fold) and miR-181b (1.5-fold) (data not shown). RT-PCR was performed to validate the microarray data in 20 paired HCC/nontumor cases (Suppl. Table 2). When these tissues were evaluated for HBx by staining and miR-181a by real-time RT/PCR, 11 of 15 HBx positive non-tumor liver samples had elevated miR-181a (73%) (P < 0.01) (Fig. 4). Among the corresponding tumors, 5 were HBx positive, but only one had higher levels of miR-181a compared to non-tumor. These results suggest that miR-181a is an epigenetic target of HBx both in cell lines and in liver. This suggests that EpCAM may be up-regulated by HBx targeting both β-catenin and miR-181.
HepG2X and HepG2CAT cells were tested for their ability to form spheres in 3D culture systems (clonogenic potential), to induce tumors in nude mice, and promote cell migration. It has been shown that stem/progenitor cells and CSCs can form spheroids in vitro suggesting the presence of self-renewing cells (34, 35). In this work, HBx-expressing cells formed spheroids by day 3 after inoculation. About 4-fold more spheroids were derived from HepG2X compared to HepG2CAT cells after 16 days in culture. Some of these spheroids exceeded 100μm in diameter (Fig. 5A, C). In contrast, HepG2CAT cells formed mostly disorganized clusters that became adherent, with less floating spheroids (Fig. 5B). Primary spheroids enzymatically dissociated to single cells gave rise to secondary spheroids. When anchorage independent growth was evaluated after 28 days of culture, HepG2X cells formed 81± 20.5 colonies, while HepG2CAT cells produced 37 ± 4.9 colonies (P < 0.02) (Fig. 5D). Cell migration was then examined by Matrigel invasion assay and H&E staining after 24 hrs. About 3.5-fold more HepG2X than HepG2CAT cells migrated through Matrigel basement membrane matrix (P < 0.001) (Fig. 5E). In tumorigenicity studies, tumor onset was 30 days post-injection for HepG2X cells compared to 46 days for HepG2CAT cells (P < 0.01). HepG2X also yielded larger tumors (1.6 ± 0.3 cm3) than HepG2CAT cells (0.7 ± 0.4 cm3) (P < 0.01). Thus, HepG2X cells showed more pronounced phenotypes consistent with stem cell behavior compared to HepG2CAT control cells. Hence, the phenotypic characteristics presented above were consistent with the induction of "stemness" markers by HBx.
The finding, that HBx binds to histone deacetylase 1 (HDAC1) (8) and that HDAC epigenetically regulates the expression E-cadherin (8, 24), suggests that inhibition of HDAC1 would reverse the effects of HBx on E-cadherin. To test this hypothesis, HepG2X and HepG2CAT cells were treated with the HDAC1 inhibitor, TSA, and the levels of E-cadherin evaluated over time. TSA treatment resulted in a time dependent increase in H3 acetylation by 20-fold (Fig. 6). This was accompanied by increased levels of E-cadherin in HepG2X cells (7-fold), decreased levels of wild type β-catenin (5-fold) and EpCAM (9.5-fold) with little change in HepG2CAT cells. These results show that once HBx repressed E-cadherin expression is reversed by TSA, alterations in β-catenin and EpCAM are also observed, suggesting these events are linked.
In the present study, the pluripotent stem cell transcription factors Oct-4, Nanog, and Klf-4, as well as the "stemness"-associated markers EpCAM and β-catenin, were up-regulated in HBx expressing cells (Fig. 1). These observations were validated in tumor and adjacent nontumor liver from HBV infected patients (Figs. 2–3). HBx expressing cells also demonstrated enhanced colony-formation capability and sphere-forming activity in vitro (Fig. 5), and were more tumorigenic in vivo. miRNA array analysis showed that miR-181, which targets EpCAM (16), was up-regulated in HepG2X cells, and this was validated in vivo (Fig. 4). The tight epidemiologic association between chronic HBV infection and the development of HCC (36), combined with evidence that HBx and CSCs contribute importantly to tumor development, suggest that HBV may promote hepatocarcinogenesis, in part, by triggering "stemness." This may be mediated by HBx associated transcriptional trans-regulation and altered epigenetic regulation of host gene expression (8, 37) as well as activation of cellular kinases that alter signal transduction (5, 36). Together data suggest that HBx promoted characteristics of CSCs in liver cells.
Although chronic infection often extends for decades, it is proposed that HBx promotes "stemness" most readily in liver just prior to the appearance of HCC. This is because the levels of HBx expression in the liver increase with the length of time a carrier is infected, with the highest levels of HBx expression seen in the cirrhotic liver (2, 38). As chronic infection proceeds, virus replication decreases, and HBx expression from integrated templates increases (2). During this process, HBx impacts upon the host pathways and gene targets mentioned above. In the chronically infected liver, HBx expressed from integrated templates are often truncated at their C-terminal regions. Prior work has shown that these mutants share properties with full-length HBx (4, 39), suggesting that they may also promote the appearance of "stemness," although further work with a series of mutants will need to be conducted to address this point conclusively. However, the observations that HBx promotes "stemness" may help to understand the promiscuous and pleiotrophic properties of HBx, since once "stemness" factors are activated, cell fate reprogramming can occur, even without the sustained expression of these markers. In this context, it is interesting that the strongest and most prevalent HBx staining was found in cirrhotic livers, and relatively little or none in the majority of HCC nodules (2, 38). This is consistent with the observation that HCC arises most often from cirrhotic livers, and with the notion that the HBx promotion of "stemness" in this pathological setting, is central to early stage tumor development.
The contribution of HBx to the development of "stemness" is supported by observations that Oct-4, Nanog, Klf-4 and c-myc are up-regulated in HepG2X compared to HepG2CAT cells (Fig. 1), and Oct-4 and Nanog were detectable in several HCC nodules (Fig. 2). Independent evidence showed strongly positive clusters of Oct-4 positive cells in HCC (40). Importantly, CSCs comprise a minor population of cells that re-establish the phenotypic heterogeneity in the primary tumor and exhibit self-renewing capability on serial passaging (9). Oct-4, Sox-2, Nanog and their binding partners act as key regulators of pluripotency in early mammalian development (41). These "stemness" genes are re-expressed in cancer cells, and the reactivation of these factors contributes to tumorigenesis in somatic tissues (10). For example, Oct-4 and Nanog expression was observed in seminoma, retinoblastoma, oral squamous cell carcinoma, bone sarcoma, breast and a colon cancer cell lines compared to their normal counterparts (42). It is proposed that some of these same pathways are turned on by HBx in hepatocarcinogenesis.
HBx contributes to the pathogenesis of HCC by several pathways, resulting in the sustained activation of EpCAM. Prior studies have shown that EpCAM is present on subsets of normal epithelia (bile duct epithelium), numerous tissue stem and progenitor cells (fetal hepatoblasts and hepatic stem cells) (12), and most carcinomas including HCC (43, 44). Although normal adult hepatocytes do not express EpCAM (12, 44), it is striking that HBV infected hepatocytes do (Fig. 2C), implying that HBx confers "stemness" properties on at least some infected cells. The significant co-staining between HBx and EpCAM in the nontumor liver is consistent with this hypothesis. EpCAM acts as a mitogenic signal transducer in vitro and in vivo via nuclear translocation of the EpICD, the latter of which was also observed in a few cases here (Fig. 2B). Importantly, the scaffolding protein, FHL2, which is a co-activator of β-catenin, bridges EpCAM with β-catenin and Lef-1, thus promoting interactions of EpCAM with DNA (13). Involvement of TCF/Lef-1, a major regulator of c-myc and cyclin E expression (45), may explain the ability of EpCAM to rapidly up-regulate their expression, thereby inducing cell proliferation. Importantly, EpCAM also has the ability to sustain "stemness" through EpICD binding to Oct-4, Nanog, Sox-2, and Klf-4 promoter regions, promoting the expression of these genes in human embryonic stem cells (14). The up-regulated expression of EpCAM by HBx is consistent with possibly similar events occurring in HCC.
Several studies have shown that HBx up-regulates and stabilizes β-catenin (Fig. 1) (20, 30). β-catenin transcriptionally up-regulates EpCAM via the presence of two Tcf binding elements in the EpCAM promoter (15). Nuclear accumulation of β-catenin induced EpCAM gene expression in cultured human hepatocytes and HCC cell lines (15). In tumor xenograft transplantation models, only EpCAM-positive cells efficiently initiated the development of invasive tumors, even after serial transplantation, demonstrating that EpCAM-positive cells display CSC-like characteristics (43). Thus, the HBx up-regulation of β-catenin (Fig. 1), and β-catenin mediated induction of EpCAM, may be critical in initiating and maintaining CSCs growth.
E-cadherin, which is important for the maintenance of cell polarity and the structural integrity in tissue, is down-regulated by HBx (24). Abnormalities in the expression and cellular distribution of E-cadherin are frequently associated with de-differentiation and invasiveness in a variety of human malignancies, including HCC (23). HBx has been found to repress E-cadherin expression at the transcriptional level through hypermethylation of the E-cadherin promoter (24), and possibly by alterations in HDAC function following complex formation with HBx (8). The latter is supported by evidence that TSA, an inhibitor of HDAC1, relieves the suppression of E-cadherin by HBx (Fig. 6). Independent evidence has shown that the inhibition of HDAC by TSA could induce hyperacetylation of histones and restore the expression of E-cadherin (46). HBx mediated repression of E-cadherin releases β-catenin from its role in cell adhesion. Prior work has shown that HBx induced adherens junction disruption is src kinase-dependent, and resulted in the accumulation of cytoplasmic β-catenin (25, 47). In the nucleus,β-catenin binds Tcf/Lef factor and acts as a transcriptional activator of growth-regulatory genes including those involved in self-renewal of stem cells. Studies have revealed that Tcf3 factor co-occupies almost all promoter regions occupied by stem cell specific transcription factors, including Oct-4 and Nanog, and this suggests the mechanism how β-catenin regulates the expression of "stemness" genes (17, 18). Hence, the finding that HBx constitutively activated wild type β-catenin in up to 80% of HCCs (20) underscores the potentially close relationship between HBx, activated wild type β-catenin, and stem cell renewal.
Recent studies show that altered expression of specific miRNAs is involved in tumorigenesis (33). Microarray data from HBV associated HCC and nontumor liver tissues showed that highly conserved miR-181 family members were over-expressed in HSCs and CSCs and thus contributed to the maintenance of "stemness." Forced miR-181 expression enriched EpCAM+ HCC cells with stem cell properties, whereas miR-181 blockage induced differentiation.16 Interestingly, data from this study showed elevated miR-181 expression in HepG2X cells compared with HepG2CAT cells, and in most HBx positive tumor and nontumor samples (Fig. 4), suggesting that miR-181 is an epigenetic target of HBx. Collectively, these data suggest that HBx may contribute to HCC by promoting the development of "stemness."
Financial support. This work was supported by grants CA104025 and CA111427 awarded to Dr. Feitelson.
The authors would like to thank Drs. Yongwen Chen and Cheng-ying Yang from the Third Military Medical University for the paraffin blocks of tissue, and Dr. Eugeney Bichenkov for assistance with the figures.
Conflict of interest statement: none declared.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information 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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The curvature of this teak stool blends form with function for an outstanding experience in comfort and relaxation as it cradles you in ergonomic design. The micro-smooth finish provides worry free use as a teak shower stool while it invites your imagination for other uses. This teak stool will support 325 pounds in the seated position. Our teak stool is made from eco-friendly teak from renewable plantations and is suitable for residential and commercial applications.
As you browse through our pages and speak with our knowledgeable customer representatives, you will find there are quite a few reasons why many have returned and recommended others to shop with us over the past 12 years. Here are some that we often hear why many of our customers think we stand out:
The furniture was well packed and arrived quickly and in excellent condition. It is beautiful and comfortable. We are very pleased with our purchase. We would definitely purchase from you again. Thank you for offering a quality product, and excellent service.
C M , Aurora , IL
Assembly Service Notice
Assembly is included with this item when this option is selected. With the assembly option selected, orders that are $1500 or over will also include White Glove Service. White Glove Service, in addition to assembly, includes unpacking, placement of furniture as well as removal and disposal of all debris including cartons and wood crating material. In stock cushions and in stock umbrella fabrics will be included with Assembly/White Glove Services. Made to order cushions and made to order umbrella fabrics are shipped separately via UPS and are not included with Assembly/White Glove Services.
Please allow 2-4 weeks for delivery when choosing Assembly/White Glove Services. Standard, non-assembled, delivery is 2-10 days.
Teak Treatment Service Notice


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The Canon EOS 650D Digital Camera is an ideal camera for any level photographer; combining both high quality imaging power and a wealth of functions that enable you to create beautiful imagery with ease. This 18.0 megapixel DSLR features an APS-C-sized CMOS sensor and a DIGIC 5 image processor for fast operation and tremendous low light performance to an expanded ISO of 25600. A quickened 5.0fps continuous shooting ability and fast 9-point cross-type AF system are also supported by the performance of the DIGIC 5 processor. This combination of technologies also results in tremendous image quality with smooth color gradations and clarity for both images and videos
Full HD 1080p video is supported and features a full-time Movie Servo AF system for continuous auto focusing while using live view to record movies. When using one of Canon's STM lenses, ultra-quiet and smooth performance is possible for fluid HD movie making. Additionally full manual sound and exposure control is supported, giving you a wide range of controls for handling your videos. For monitoring of videos and still imaging, a 3.0" Vari-angle touch screen LCD is incorporated and flips out and tilts for viewing from a wide variety of angles. The touch screen operation is highly intuitive and even allows you to control your focus points from the LCD.
The Scene Intelligent Auto Mode combines the essential auto modes with an intelligent EOS Scene Detection System to quickly and automatically determine the situation type you are working in and apply the necessary adjustments to render the scene correctly. Once the image has been captured, it will also apply enhancing techniques in order to improve skin tones and overall image coloration. Other creative modes include an HDR Backlight Control mode for improving the effective exposure value range of your image; a Handheld Night Scene mode which utilizes multi shot noise reduction techniques to give you improved low-light performance without the noise of high ISOs; and seven different creative filters to apply unique effects directly to your images in-camera.
The 650D is compatible with SD/SDHC/SDXC memory cards and Canon's full line of EF and EF-S lenses.
**Power cord may not fit your country , we will provide FREE charger adaptor if necessary**
|Camera Type
|Digital SLR with Interchangeable lenses
|Lens Mount
|Canon EF
|Camera Format
|Canon APS-C (1.6x Crop Factor)
|Effective Pixels: 18.0 Megapixels
Max Resolution: 17.9 MP: 5184 x 3456
|Sensor Type / Size
|CMOS, 22.3 x 14.9 mm
|File Formats
|Still Images: JPEG, RAW
Movies: MOV
Audio: Linear PCM
|Bit Depth
|Noise Reduction
|Memory Card Type
|AV Recording
|Video Recording
|Yes, NTSC/PAL
|Aspect Ratio
|3:2, 16:9
|Video Clip Length
|Up to 8GB or 22 Min
|Audio Recording
|With Video, Stereo
|Focus Control
|Focus Type
|Auto & Manual
|Focus Mode
|Single-servo AF (S), Continuous-servo AF (C), Manual Focus (M)
|Autofocus Points
|Viewfinder Type
|Viewfinder Coverage
|Viewfinder Magnification
|Approx. 0.85x
|Diopter Adjustment
|- 3.0 to +1.0 m
|Display Screen
|3.0" Rear Touchscreen LCD (1040000)
|Screen Coverage
|Live View
|Exposure Control
|ISO Sensitivity
|Auto, 100-12800 (High Sensitivity Mode: 100-25600)
|Type: Electronic & Mechanical
Speed: 30 - 1/4000 sec
|Remote Control
|RC-1, RC-5, or RC-6 (Optional)
|Metering Method
|Spot metering, Center-weighted average metering
|Exposure Modes
|Modes: AE Lock, Aperture Priority, Manual, Program, Programmed Auto, Shutter Priority
Compensation: -5 EV to +5 EV (in 1/3 EV steps)
|White Balance Modes
|Auto, Cloudy, Custom, Daylight, Flash, Fluorescent (White), Kelvin, Shade, Tungsten
|Built-in Flash
|Yes: Auto, FE Lock, Off
|Max Sync Speed
|1 / 200 sec
|Continuous Shooting
|Up to 5.0 fps
|Dedicated Flash System
|External Flash Connection
|Hot Shoe, PC Terminal
|Shutter Lag
|0.075 Seconds
|Self Timer
|10 sec, 2 sec
|AV Output, HDMI C (Mini), USB 2.0
|Wi-Fi Capable (With Optional Transmitter)
|1x LP-E8 Rechargeable Lithium-Ion Battery Pack, 7.2VDC, 1120mAh
|AC Power Adapter
|ACK-E8 (Optional)
|Operating/Storage Temperature
32 to 104 °F (0 to 40 °C)
Humidity: 0 - 85%
|Dimensions (WxHxD)
|5.2 x 3.9 x 3.1" / 13.21 x 9.91 x 7.87 cm
|1.14 lb / 0.52 kg
AC-Electronic.com Offers 12 Months Free Seller Warranty for all products purchased in AC-Electronic.com(B2B order is excluded).
It warrants products to be free from defects in material and workmanship during warranty period. If the product proves to be defective in material or workmanship during the warranty period, AC Electronic Limited will, at its sole option, repair or replace the product with a same or similar product depends on stock. And all products must be sent back to AC Electronic Limited in Hong Kong for warranty.
Please click HERE to read full warranty terms.
Further more, AC-Electronic.com also offer 7 DAYS EXCHANGE for all products purchased in AC-Electronic.com, so, no worry about :
Please click HERE to read full Retun terms.


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How to repurpose a soda bottle to pick fruit
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Create your own Cooling Cinnamon Body Powder to help alleviate sticky heat rash.


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How 'The Dark Knight Rises' Should Have Ended
"How 'The Dark Knight Rises' Should Have Ended," by HISHE, is more like "Why 'The Dark Knight Rises' Sucks": the deep, growly Batman voice; the fact that everyone knows Batman's identity, no one shoots Bane when he's ripe for shooting, and Comm. Gordon sends all of Gotham's police force underground at the same time. Not to mention Talia's death, which has to be one of the lamest death scenes by an actress who's won an Academy Award. (Come on, Marion. It's time that we began to laugh and cry and cry and laugh about it all again.)
Here it is:
My own review of "Dark Knight Rises" raises similar, if less humorous points.
Tags: The Dark Knight Rises, Batman, HISHE, Christian Bale, Christopher Nolan, Joseph Gordon-Levitt, Marion Cotillard, Anne HathawayPosted at 10:38 AM on Sun. Aug 26, 2012 in category Batman
Twitter: @ErikLundegaardTweets by @ErikLundegaard
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Product Information
Potter Waltz by London Symphony Orchestra - Digital Sheet Music
At a Glance
Potter Waltz digital sheet music. Contains printable sheet music plus an interactive, downloadable digital sheet music file.
* This product is available worldwide
Arrangement Details
The Arrangement Details Tab gives you detailed information about this particular arrangement of Potter Waltz - not necessarily the song.
Song Details
The Song Details Tab gives you detailed information about this song, Potter Waltz.
Related Products
The Related Products shows you other products that you may also like, if you like Potter Waltz.
There are No Reviews written for "Potter Waltz"
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Musculoskeletal System III
This course is designed to provide the student with the necessary skills for prevention, examination, evaluation, and intervention of musculoskeletal disorders of the spine and TM joints. It is the third in a series of musculoskeletal based classes and will complete the required musculoskeletal content of the PT program. The student will learn an evidence-based and systems approach to patient care. Specifically, this course will teach the student how to determine which impairments are related to specific functional limitations and disabilities of the individual with spinal disorders. It will consider spinal impairments throughout the life span as well as throughout the natural history of the problem. An eclectic approach to interventions will be stressed along with current literature findings.
Note that this information may change at any time. Please visit the Student Link for the most up-to-date course information.


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Jog Falls Hotels
10 Jog Falls Hotels List ranked by travellers
Average Rating - 5.00/7, based on 229 Ratings & 129 Reviews | Price Range : Rs. 100 - 3000
4 Photos
Rank 1
Garden Land, Sirsi, Uttara Kannada
"Food served by Shivani Hotel is awesome and service is excellent. Any one plan to trip to Jog..." More ›Hari, Bangalore, 2 days ago
8 Photos
Rank 2
Jog Falls, Sagar Taluk
"Excellent view from hotel available with both AC and non AC rooms. Rooms are very clean, food and..." More ›Rahul, Bangalore, 5 days ago
4 Photos
Rank 3
Aralagodu Village, Near Jog Falls, Sagara Taluka,
"In Gundimane I have stayed for about 3 days. The location of Gundimane is excellent! Rather than..." More ›Varun, Salem, 3 months ago
1 Photos
Rank 4
Tumri Village, Sagara Taluk, Haalkere
"A nice hotel. Decent rooms and a good ambiance. The staff and services were quite good. Overall,..." More ›Sai, Hyderabad, Jul 24, 2014
Rank 5
Village Nandigodu, Honnesara Post, near Genasinakuni, Heggodu Gram Panchayat, Sagar Taluk
"Its a quite a good experience to visit the place. We enjoyed the peaceful presense in the thick..." More ›Srikrishna, Bangalore, Aug 31, 2012
India Says Yes Award
Winner for categories:
Rank 6
"Very good home stay near Jog. Good location and good homely food. Good cottage and clean..." More ›Ravikumar, Bangalore, 3 months ago
Rank 7
Yellapur Road, Sirsi Taluk, Uttara Kannada
"We have been to Jog falls in Shimoga district to enjoy the water falls. We booked the hotel for..." More ›Vino, Chennai, Nov 4, 2014
Rank 8
Urugallu Village, Near Biligaru, Kargal - Bhatkal road-Sagar taluk
"The hotel was nice.The people handling this place were very kind and nice and the place was..." More ›Nagaraj, Bangalore, Feb 17, 2012
Rank 9
Urugallu Village, Near Biligaru, Kargal - Bhatkal Road
"the hotel was in pleasent place. it was good to stay.we went with group of team, and the staff..." More ›Lakpathi, Bangalore, Aug 28, 2013
Rank 10
Near Water Falls, Jogfalls, Shimoga Dist.
"I had tried booking at this hotel through the Youth hostel helpline for bookings.. not sure what..." More ›Shilpz, Mumbai, Aug 25, 2013
Hotels in Jog Falls with Tariff Rates
- There are 10 hotels in Jog Falls reviewed by actual travellers. The top rated Jog Falls hotels are Haalkere Homes, GundiMane Home Stay, Matthuga�Home Stay. The most sought after hotels, based on these reviews are Group stay hotels, Couple stay hotels. The lowest available tariff for Jog Falls hotels stands at Rs.100 and highest hotels rate is approximately Rs.3000. The cheapest Jog Falls hotel deals are available at Rs.600. You can search for Jog Falls Home Stay(5), Jog Falls B&B/Guest house/Hostel/Lodge(3), Jog Falls Hotel
There are different types of hotels in Jog Falls available for families, lovers, kids, friends and specially for newly married couples who are looking for perfect honeymoon destination.
Hotels in Jog Falls by PriceThere are 8 Budget hotels in Jog Falls which are priced below Rs. 2500. Tariff for hotels in Jog Falls starts from 100. Cheap hotels in Jog Falls may provide basic amenities like a private bedroom, colour TV, Telephone, AC, 24-hour front desk and housekeeping staff on call. These hotels are affordable and value for money.
Hotels near Main Attractions in Jog FallsThe most popular attractions in Jog Falls are Tunga Anicut Dam, Sharavati River Jog Falls, Thyvare Koppa Lion & Tiger Reserve, Linganmakki Dam and BRP Dam.
Hotels near Main Transportation PlacesThere are 1 railway station in Jog Falls which is Shimoga railway station. You can find 19 hotels near Shimoga railway station.


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PRLog - Nov. 9, 2009 - COLOGNE, Germany -- One of the major factors for the growth of GPS is increase in the demand of mobile location technologies. Further, timing and location capabilities of GPS account for its growth in applications such as disaster relief & emergency services, banking, and power grid control. The automotive and consumer industries have a large demand for GPS.
GPS is evolving from being a product to being a feature; and amidst recession, companies are making use of GPS for self monitoring. For example, distribution, shipping and construction companies can reduce operational and fuel costs of fleet vehicle by implementing GPS tracking solutions.
With the increasing demand for GPS solutions in the consumer segment, customers prefer to purchase from traditional CE retail channel, including online merchants.
"The demand for navigation devices is increasing due to a higher awareness towards positioning technology, decreased prices of navigation devices, enhanced user friendly features, and ease of access," says one of the Infiniti Research experts, "the Global GPS market is expected to reach $69,658 million by 2012 from 27,470 million in 2008; growing at a CAGR of 26 percent."
The report The Global Positioning System Market 2008-2012 forecasts the market size of GPS over the period 2008-2012. The total GPS market is segmented into various geographic regions (representing the revenue from GPS in each of these regions). The report also discusses about the market trends, sales drivers and challenges that characterize the GPS market and profiles some of the key vendors of this market.
Infiniti Research: The Global Positioning System Market 2008-2012:
Infiniti Research: More market data and market reports:
# # #
markt-studie.de, founded in 2002 has emerged as a leading online portal for market surveys and market research in German speaking areas. Four years later the English language portal reports-research.com was introduced due to the extraordinary success of the portal. Again one year later estudio-mercado.es - the Spanish spoken portal - was founded. The objective of the three portals is to competently and efficiently support consultants and decision makers in management, sales and marketing in the search for worldwide market research. Prospective buyers can look into more than 60,000 market market market surveys from more than 200 international publishers, current market data for more than 6,000 branches worldwide, 10,000 company profiles as well as a free-of-charge research and recommendation service for individual market research.


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I am looking for the best way to pipe data from one stream to another without having to deal with buffering myself. Basically the equivalent of the node.js stream.pipe function.
There is stream_copy_to_stream, but according to the comments on the manual page it is quite a memory hog (possibly reading the whole stream into memory?).
Of course I could do something like this:
$fh = fopen('filename.txt', 'rb');
$out = STDOUT;
while (!feof($fh)) {
$bytes = 1024;
fwrite(STDOUT, fread($fh, $bytes), $bytes);
But I'm hoping there is an easier API to accomplish this, without having to do the buffering myself.


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15 November 2011
The Bricklayers Arms has won the Bromley Best Bar None Award for Responsible Retailing (small venue) 2011, thanks to its exceptionally high standards of innkeeping.The scheme, run in conjunction with Bromley Council and the police, aims to promote the borough as a safe and enjoyable place to live, work and socialise.
Read more
31 October 2011
Ale drinkers are being challenged to try something spooky this Halloween – if they dare.The eerily tasty Spooks Ale from Shepherd Neame will be available in bottles and cask from mid-October.
28 October 2011
Shepherd Neame Ltd, the Kent-based brewer and pub operator, announces the appointment of Mark Rider (35) as Finance and IT Director. Mark will join the company in January 2012 and will replace Ken Littlefair on the Board upon his retirement in March 2012.
21 October 2011
Shepherd Neame has further boosted its expertise in beer with the appointment of Richard Frost as head brewer.Mr Frost – currently head brewer at Banks's Brewery in Wolverhampton – brings a wealth of industry knowledge and expertise to an already highly experienced team.
18 October 2011
Shepherd Neame's new events venue, The Old Brewery Store, has a packed programme of entertainment throughout November and December.
Contact us by email
Contact us by phone
Shepherd Neame LimitedThe Faversham Brewery17 Court StreetFavershamKent. ME13 7AXUnited Kingdom


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Quiver Inn 2
From $135 / night
Description from the manager
Description from the manager
Bedrooms: 2/
Bathrooms: 2/
Sleeps: 4/
Type: House
One side of a duplex, Quiver Inn 1 is a cozy and comfortable vacation home perfect for couples or a small family. It offers 2 bedrooms and 1 bathroom on the upper level and the kitchen, dining, half bathroom and living room on the main level. The kitchen and dining area are open and bright and lead into the living room. The living room has a stone fireplace, comfortable furniture and patio doors leading out to the back deck and shared outdoor hot tub. The hot tub is Winter Only. This unit has A/C and WIFI is available throughout. This quaint duplex is located just one block from the main ski lodge. A free shuttle is offered during the ski season....Read more
About the manager
About the manager
Big Powderhorn Lodging Association
Big Powderhorn Lodging Association
- Response rate: 100%
- Response time: A few hours
- Calendar updated: today
Overall rating:
4.3 / 5.0
Quiver Inn 2 Ironwood, Michigan
Additional Location Information
Show rates details
Also consider
- FlipKey has rentals in over 11,000 destinations around the world:
- Port Aransas vacation rentals - 554 rentals from $133/night
- Broken Bow cabins - 15 rentals from $141/night
- Blue Ridge cabin rentals - 264 rentals from $100/night
- Maine vacation rentals - 2,245 rentals from $100/night
- Eureka Springs cabins - 81 rentals from $95/night


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Auction price: Register or sign-in to see quarterly averages
or visit the Wine Market Journal
Drink between: 2014 - 2033 (Add My Dates)
312 Tasting Notes
3 Tasting Notes
585 Tasting Notes
Holy cow! How to rate this? First describe it... Deep sweet blackberry and cherry nose with a hint of mint freshness and some Bordeaux like elements. Amazingly well balanced palate. Acidity, sweetness, bitterness and alcohol all perfectly together. Sweet and fruity with depth and layered. Amazing finish which never seems to end. Exeptional wine. One of the best I have ever tasted, absolutely sublime! And therefore a well deserved 99 points.
Très riche et dense, fort complexe et jeune.
Texture juteuse et bonne acidité, un brin moderne mais bien avec une belle pièce de viande.
My heaven in a bottle. Always perfect. Earthy and enough muted fruit to go well with a steak!
Attractive enough, but not really soaring at this point of its development
First of these 2007 CDPs from BPW that doesn't smell of oxidized, stewed fruits. Nose leads with earth, smoke and herbs followed by ripe red and dark fruit. Concentrated and long, but with structure. Runs a little hot, but really enjoyable overall.


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Messages List
Re: US Passports and Certificate of Citizenships for Americans abroaKaren, this is very helpful! Unfortunately, the email cut off above. Can you try it again? Or send her email to me? My email is jjtell89@... May I ask
Jul 3#9896
Re: US Passports and Certificate of Citizenships for Americans abroaKaren, that is very helpful! I am hopeful she will be given an IH-3 visa so this won't be an issue. She will be legally adopted in Serbia, not guardianship. I
Jun 15#9895
Re: IH-3 Visa, Passports, CoC, ectHello Randy, I think that you meant this info for my situation: American citizen living in UK; married to Uk citizen; adopted a 3 year old and 6 year old in
Sean Gates
Jun 15#9894
Fetching Sponsored Content...
Re: IH-3 Visa, Passports, CoC, ectOne last thing. You cannot get a US passport for your child unless you have an immigrant visa. What I suggest is that since you have your child with you abroad
Randy Barlow
Jun 15#9893
Re: US Passports and Certificate of Citizenships for Americans abroaDon't know what an i-800 is but: This lawyer in Vancouver, Washington helped me get an American adoption and a US passport in about a week without any wait
Karen Greene
Jun 13#9892
Re: IH-3 Visa, Passports, CoC, ectThank you all for the responses! It seems like the best course of action is to fly to the U.S. first and wait to see what happens. All this does help give a
Jun 13#9891
Re: IH-3 Visa, Passports, CoC, ectWe used the N600K for COC as expats returning abroad following a (Hague) adoption from China that is final in China at time of adoption. Lisa
Jun 13#9890
Re: IH-3 Visa, Passports, CoC, ectHello Debbie, Thanks for your reply. We adopted via the UK adoption process, so we had a British homestudy. I can check with the U.S. Embassy in London, if I
Sean Gates
Jun 12#9889
IH-3 Visa, Passports, CoC, ectHello Elaine, From what you have written below, it is unclear to me which adoption process you followed in order to adopt your children from Russia in 2007
Debbie DriessenGrika
Jun 12#9888
Re: IH-3 Visa, Passports, CoC, ectHello, My British husband and I (American) adopted our two children from Russia in 2007, before the Hague Convention was in effect. Subsequently, we
Sean Gates
Jun 12#9887
Re: IH-3 Visa, Passports, CoC, ectI didn't read the previous threads but what happened to Marty is also what happened to us. Sonya Sorry for any mistakes as I sent this from my phone.
Sonya Todd
Jun 11#9886
Re: IH-3 Visa, Passports, CoC, ectHi Marty, Am pretty sure that was because to the DHS, you live abroad and maybe you did not have a US address with your Adoption agency OR you have to prove
Charbel Bousraih
Jun 11#9885
Re: IH-3 Visa, Passports, CoC, ectHey everybody, don't mean to be a downer, but we had a very different experience from what you all describe. We adopted our Thai daughter through Hague process
Jun 11#9884
Re: IH-3 Visa, Passports, CoC, ectHi Pinkpuffy, I'd like to add the suggestion that you (sooner rather than later, if only because later you'll have still more tasks staking a claim on your
Barbara Pines
Jun 10#9883
IH-3 Visa, Passports, CoC, ectJust to add to this conversation: If your adoption is finalized in Serbia and you have seen the child prior to adopting him/her, then you will be issued the
Debbie DriessenGrika
Jun 10#9882
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Category:Regions of Central America
From Wikipedia, the free encyclopedia
This category has only the following subcategory.
- ► Central America Volcanic Arc (7 C, 4 P)
Pages in category "Regions of Central America"
The following 6 pages are in this category, out of 6 total. This list may not reflect recent changes (learn more).


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Montreal, QC (Sports Network) - James Reimer made 37 saves for his seventh career shutout and the Toronto Maple Leafs used a well-balanced offensive attack in a 6-0 rout of the Montreal Canadiens on Saturday.
Phil Kessel had a goal and two assists for the Maple Leafs, who have won three in a row for the first time since Feb. 1-6 of last season. James van Riemsdyk and Dion Phaneuf each added a goal and an assist, while Leo Komarov, Tyler Bozak and Korbinian Holzer also lit the lamp.
"You guys have been asking me about it for a while, but he's a special player," Phaneuf said of Kessel. "He's scored a lot of goals for a while, consistently. Sometimes they don't go in, but now that they're going in he's a guy who can get hot, so it's nice to see him get another one tonight."
Toronto improved to 2-0 against the Canadiens this season. The Leafs won at Montreal by a 2-1 score on Jan. 19.
Carey Price stopped 21-of-27 shots for the Canadiens, who have lost three straight.
The Maple Leafs took control 59 seconds into the game. Nikolai Kulemin stole the puck from Montreal's Andrei Markov at the blue line and went racing down the right wing. Kulemin slid a pass to Komarov, who put the puck into the back of the net.
Bozak lit the lamp at the 6:33 mark to make it 2-0. After winning a faceoff in the offensive zone, Bozak made his way to the front of the net. A shot from the left point ended up on the stick of Bozak, who knocked in the rebound.
"We had a really, really slow start and they beat us with speed," Montreal coach Michel Therrien said. "They were ready to play and we were not ready to play."
A quick goal by Toronto in the early stages of the second stanza made it 3-0. Kessel controlled the puck behind the net before finding van Riemsdyk in front.
Kessel's power-play goal with 1:59 remaining turned the game into a rout. His shot from the top of the left circle got past Price.
Reimer was sensational in the middle frame, making 20 saves.
Holzer and Phaneuf scored in the third period to account for the final margin.
Toronto has won five of its last six at Montreal ... Canadiens forward Travis
Moen returned to the lineup after missing Thursday's contest with a lower-body
injury ... Toronto went 2-for-6 on the power play, while Montreal went 0-
for-4 ... The Maple Leafs finished their three-game road trip with a perfect
Former Heisman Trophy winners Matt Leinart, Eric Crouch and Rashaan Salaam are among 76 Football Bowl Subdivision players on the ballot for election into the 2016 class of the College Football Hall of Fame.
The regular season in the NASCAR Sprint Cup Series has reached its midpoint, with 13 races completed and 13 to go before the championship Chase begins.
The Houston Texans have signed defensive lineman Brandon Deaderick, who played last season with the New Orleans Saints.
The Toronto Blue Jays activated catcher Dioner Navarro from the 15-day disabled list following a 2-0 loss to the Washington Nationals in the opener of Tuesday's doubleheader.
Sacramento Kings forward Carl Landry underwent surgery on Tuesday to repair a torn ligament in his right wrist, the team said.
The Montreal Canadiens revealed on Tuesday that defenseman Jeff Petry has agreed to terms on a six-year contract extension.


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JLM Commercial Advisors announced that Mary Jo Giammalva has joined the firm as Sr. Vice President. After spending the last 14 years at Weingarten leasing their 6.5 million SF Houston Industrial portfolio, Giammalva will partner with President Lane Guinn to focus on corporate services, representing end-users of industrial and office properties.
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New Full-Text Catalog (General Page)
SQL Server 2016
Topic Status: Some information in this topic is preview and subject to change in future releases. Preview information describes new features or changes to existing features in Microsoft SQL Server 2016 Community Technology Preview 2 (CTP2).
Use this dialog box to create a new full-text catalog.
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Summary of findings [Explanations]
The incidence of epilepsy following supratentorial craniotomy for non-traumatic pathology has been estimated to be 15% to 20% (Foy 1981); however, due to the nature of the underlying disease for which surgery is undertaken, the risk of post-craniotomy seizures may vary from 3% to 92% over a five-year period. It is likely that such seizures may cause epilepsy in previously unaffected people. The probability of de novo seizures occurring in people who have no history of epilepsy decreases over time. The highest incidence of postoperative epilepsy (two-thirds of the seizures) occurs within the first month after cranial surgery (North 1983a), and 75% of those who do develop epilepsy will do so within one year of surgery. Few people (approximately 8%) have their first seizure more than two years after surgery. The risk of seizures for particular groups of people is higher; for example for those with an arteriovenous malformation who have had a spontaneous intracerebral haematoma, the overall risk does not fall below 10% between year two and five after surgery, while people who suffered from an abscess continue to run a risk of developing epilepsy (92%) after five years (Shaw 1991).
Due to the risk of postoperative seizures, the prophylactic use of antiepileptic drugs (AEDs) has been advocated for patients undergoing cranial surgery. Uncontrolled retrospective trials support the use of AED treatment in patients with a predisposition towards developing postoperative seizures (Matthew 1980) and data from pathological trials suggest that certain AEDs could have a neuro-protective action on damaged cerebral cortex (Calabresi 2003).
However, it is also argued that AEDs should not be used prophylactically, but should only be administered following at least one seizure (Temkin 2002). Other investigators maintain that early postoperative seizures do not justify the diagnosis of epilepsy and only late seizures are considered to be true epilepsy (Manaka 2003).
To inform decision-making regarding the prophylactic use of AEDs for people undergoing craniotomy, reliable, high-quality evidence is required. Benefits and harms and any trade-offs between these need to be examined carefully. Potential benefits include reduced short-term seizure recurrence, reduced long-term epilepsy rates, and better surgical outcome and quality of life. Harms include adverse effects and poorer surgical outcome. This review will provide a summary of the currently available evidence from randomised controlled trials (RCTs) regarding the prophylactic use of AEDs for people undergoing craniotomy by examining the following outcomes: occurrence of early and late seizures, occurrence of death, functional disability and occurrence of adverse events.
To determine the efficacy and safety of AEDs when used prophylactically in people undergoing craniotomy for all pathologies.
Criteria for considering studies for this review
Types of studies
- RCTs, in which an adequate method of randomisation and allocation concealment was used.
- Double, single or unblinded trials.
- Placebo (PCB)-controlled, active drug control group or no treatment control group.
Types of participants
People of any age and either gender undergoing a supratentorial or infratentorial craniotomy for either therapeutic or diagnostic reasons for all pathologies, who have had no history of seizures or prior exposure to AEDs.
Types of interventions
- The active treatment group receive treatment with any AED administered prior to or immediately post craniotomy.
- The control group receive matched PCB, different AED or no treatment.
Types of outcome measures
Primary outcomes
(1) Early seizures
The proportion of people experiencing seizures occurring within the first week following craniotomy.
(2) Late seizures
The proportion of people experiencing seizures after the first week from craniotomy including follow-up period of one, two and five years postoperatively from craniotomy.
Secondary outcomes
(1) Death
The proportions of deaths occurring within the treatment period or during follow-up.
(2) Functional outcome
The proportion of people experiencing disability (partially or fully dependent on others in normal activities of daily living).
(3) Adverse effects
The proportion of people who experience any of the following adverse events:
- skin irritation;
- dizziness;
- fatigue;
- nausea;
- headache.
In addition, we decided to look at the proportion of people experiencing the five most common adverse effects mentioned in the included trials if these differed from the list above.
Search methods for identification of studies
See Appendix 1 and Appendix 2 for search strategy used.
Electronic searches
We searched:
- the Cochrane Epilepsy Group's Specialized Register (19 September 2012);
- the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library Issue 9, 2012) using the search strategy outlined in Appendix 1;
- MEDLINE (Ovid, 1946 to September week 1, 2012) using the search strategy outlined in Appendix 2.
No language restrictions were imposed.
Searching other resources
We reviewed the reference lists of retrieved trials to check for additional reports of relevant studies.
Data collection and analysis
Selection of studies
Two review authors (JP and JG) independently assessed articles for inclusion. Any disagreements were resolved through mutual discussion, failing this a third-party (AM) opinion was sought. The same review authors independently carried out data extraction and assessed risk of bias. Again, disagreements were resolved by mutual discussion, failing this a third-party opinion (AM) was sought.
Data extraction and management
The following information was extracted for each trial using a data extraction sheet.
Methodology/trial design
- Method of randomisation and concealment.
- Method of blinding.
- Number of people excluded from analyses.
- Duration of baseline, treatment and follow-up periods.
- Type of AED and dose tested.
- Time of treatment commencement.
Participant demographics
- Total number of people randomised to each group.
- Age/gender.
- Pathological group.
- Type of surgery.
- Site of lesion.
- Number of people with previous acute symptomatic seizures.
- Sample size.
- Summary data for each intervention.
For all trials we attempted to confirm the above information with trial authors/researchers and sponsors.
Assessment of risk of bias in included studies
Two review authors (JP and JG) independently made an assessment of risk of bias for each trial using the Cochrane 'Risk of bias' table as described in Higgins 2002. Any disagreements were discussed and resolved. Included trials were rated as adequate, inadequate or unclear on six domains applicable to RCTs:- randomisation method, allocation concealment, blinding methods, incomplete outcome data, selective outcome reporting and other sources of bias. A 'Summary of findings' table was created; however, the GRADE approach for determining quality of evidence was not assessed as no trials were combined.
Measures of treatment effect
Treatment effects are presented as they were in the original reports.
Assessment of heterogeneity
Clinical heterogeneity was assessed by examining the differences in trial characteristics in order to inform decisions regarding the combination of trial data.
Assessment of reporting biases
Reporting biases, such as publication bias, were examined by identifying certain aspects of each trial (e.g. sponsors of the research, research teams involved).
Data synthesis
Data were synthesised in a narrative format as meta-analysis was considered to be inappropriate given the differences across trials in AED treatment, trial intervention characteristics and control groups (see Table 1). Data under each comparison listed below were minimal and could not be combined across all outcomes.
Comparisons discussed in the narrative included:
- treatment group vs. control group on early seizures;
- treatment group vs. control group on late seizures;
- treatment group vs. control group on number of deaths;
- treatment group vs. control group on functional outcome;
- treatment group vs. control group on adverse effects (for each adverse effect see Types of outcome measures).
Each comparison was stratified by type of drug and control group (i.e. PCB, other AED or no treatment).
Description of studies
See: Characteristics of included studies; Characteristics of excluded studies; Characteristics of studies awaiting classification.
The search revealed 86 records identified from the databases outlined in Electronic searches. Ten further records were identified through the reference lists of included trials. Fifty-seven records remained after duplicates were removed, all were screened for inclusion in the review. Thirty-three were excluded at this point leaving 24 full-text articles to be assessed for eligibility. Following this, 15 texts were excluded (see Figure 1 and Characteristics of excluded studies for reasons of exclusion). Six trials from eight reports were included in a qualitative synthesis.
|Figure 1. Study flow diagram.
Six parallel RCTs were found (Beenen 1999; Foy 1992; Franceschetti 1990; Lee 1989; Nakamura 1999; North 1983) examining the effectiveness of AEDs on post-craniotomy seizures. The treatment periods varied across trials from three days to 24 months; in one trial the treatment period was unclear (Franceschetti 1990). People were excluded from five of the trials if they were taking AEDs already and if they had a history of epilepsy (Beenen 1999; Foy 1992; Lee 1989; Nakamura 1999; North 1983). One trial (Franceschetti 1990) included both people who had preoperative seizures (Group A) and those who did not (Group B); Group A and Group B were analysed separately compared to controls. Only data pertaining to Group B were extracted to be included within this review as they did not meet the inclusion criteria for the review.
The trial reported by Beenen et al (Beenen 1999) was a single-centre trial with a treatment period of 12 months. People aged between 18 and 80 years who were undergoing supratentorial craniotomy were eligible to be randomised. One hundred patients were randomised, 50 to phenytoin 100 mg (PHT) and 50 to valproate 500 mg (VAL) both administered intravenously immediately post operation in a recovery room. Outcomes reported included early and late seizures, death and adverse effects. No data were reported for functional outcome.
The trial reported by Foy et al (Foy 1992) was a single-centre, head-to-head (active drug comparison) trial with a treatment phase of either six or 24 months, follow-up occurred for a minimum of three years to a maximum of eight years. People aged over 16 years undergoing supratentorial craniotomy were eligible to be randomised. Two hundred and seventy-six patients were randomised, 50 to carbamazepine (CBZ) for a six-month treatment period, 56 to CBZ for a 24-month treatment period, 55 to PHT for a six-month treatment period, 56 to PHT for a 24-month treatment period and 59 to no treatment. Administration of CBZ (200 mg) was 6 hourly for the 24 hours immediately pre-operation and eight hourly thereafter; PHT (15 mg/kg) was administered 24 hours pre-operation and increased to 100 mg eight hourly thereafter. Outcomes reported included number of patients with seizures and death. No differentiation was made between early and late seizures and no data were reported for functional outcome or adverse effects. All data were reported at six months into the treatment.
The trial reported by Franceschetti et al (Franceschetti 1990) was a single-centre, head-to-head three-arm trial that included a 'no treatment' group. People undergoing surgery for supratentorial neoplasms were randomised and patients with a history of seizures formed Group A and patients who had no history of seizures formed Group B. Sixty-three Group B patients were randomised, 25 to phenobarbital (PB), 16 to PHT and 22 to no treatment. The PB (4 mg/kg) was intravenously administered daily for five days then decreased to 2 mg/kg daily via oral administration. PHT (10 mg/kg) was intravenously administered daily for five days then decreased to 5 mg/kg daily via oral administration. Outcomes reported included early and late seizures, and minimal data on adverse effects were presented.
The trial reported by Lee et al (Lee 1989) was a PCB-controlled trial with a treatment period of three days. People receiving intracranial, supratentorial surgery were eligible to take part in the trial. Four hundred patients were selected for participation and randomised, 26 early deaths occurred leaving 189 people randomised to PHT and 185 people to PCB. PHT (15 mg/kg) was administered 15 to 20 minutes prior to wound closure followed by intravenous PHT (5 to 6 mg/kg) three times daily for the first three postoperative days. Outcomes measured included number of seizures occurring within the three days of the trial. Data for late seizures, death, functional outcome and adverse effects were not recorded.
The trial reported by Nakamura et al (Nakamura 1999) was a multi-centre, head-to-head trial with a treatment phase of one year and a follow-up of two years without medication. People undergoing craniotomy for cerebral tumours, cerebrovascular disease and head trauma were selected for eligibility. Two hundred and seventy-eight patients were randomised, 141 to zonisamide (ZNS, 100 mg twice daily) and 126 to PB (40 mg twice daily). Both drugs were administered orally, at least one week before surgery and then increased (ZNS to 100 mg three or four times daily and PB to 40 mg three or four times daily) for one year followed by a tapering period of six months (three months at 100 mg (ZNS) or 40 mg (PB) twice daily then three months at 100 mg (ZNS) or 40 mg (PB) once daily). Outcomes reported were seizure frequency, death (during follow-up period only) and adverse effects. No data were collected on functional outcome.
The trial reported by North et al (North 1983) was a single-centre, PCB-controlled trial with a treatment period of 12 months. People undergoing supratentorial operation (either burr hole, craniectomy or osteoplastic flap procedures) were eligible for inclusion for the trial. Two hundred and eighty-one patients were randomised, 140 to PHT and 141 to PCB. PHT (250 mg twice daily) was administered in a recovery room intravenously, and then continued with oral medication (100 mg three times daily) for one year. Outcomes reported were early and late seizures, death and adverse effects. No data were collected on functional outcome.
Risk of bias in included studies
Out of the six trials included, only two (Beenen 1999; Foy 1992) were considered to have used adequate methods of randomisation with regards to sequence generation. The other four trials were unclear in their methods or there were no available details in text. One trial (Beenen 1999) described the use of sealed envelopes and pre-coded and packaged medication, the other five trials were rated as unclear. Four trials used adequate blinding methods by using identical treatments, one trial (Foy 1992) was unblinded and one was unclear (Franceschetti 1990). Assessments of incomplete outcome data revealed all trials to have some study attrition, one trial (Lee 1989) was unclear regarding attrition rate and analysis therefore was rated as unclear in bias on this criteria. Two trials (Beenen 1999; North 1983) reported attrition and employed intention-to-treat analysis to deal with these missing data. The other three trials were unclear as to how missing data were managed. No protocols were available for any of the trials (although these were requested from authors) therefore the completeness of reported outcome data was difficult to assess. All trials reported data outlined in the methods section of their papers and appeared to report what was set out to record. No other evidence of bias was found in five of the six trials. One trial (Foy 1992) was rated as unclear on these criteria due to the addition of a third control group after a first analysis showed few differences between groups. See Characteristics of included studies for Foy et al. See Figure 2 for 'Risk of bias' graph.
|Figure 2. Risk of bias graph: review authors' judgements about each risk of bias item presented as percentages across all included trials.
Effects of interventions
See: Summary of findings for the main comparison Antiepileptic drugs as prophylaxis for post-craniotomy seizures
Due to the variety of head-to-head drug comparisons within the included trials, the effects of the interventions will be presented by outcome measure as opposed to comparisons under trial. See Table 2 and Table 3 for individual trial results and Table 1 for a comparison of treatment protocols.
Beenen 1999 examined seizure outcome between PHT and VAL and reported 4/50 early seizures occurring in the PHT group compared to 2/50 early seizures in the VAL group. Three late seizures occurred in the PHT group compared to five late seizures in the VAL group. Foy 1992 examined three treatment groups, CBZ, PHT and no treatment. They reported 41/106 seizures (early and late) occurring in the CBZ group, 37/111 occurring in the PHT group and 25/59 occurring in the 'no treatment' group at six months. The trial by Franceschetti 1990 also looked at three groups, PB, PHT and no treatment. They reported 3/41 early seizures occurring in the PB and PHT groups and 4/22 early seizures in the 'no treatment' group. Only 39 patients were followed up and they reported 3/25 late seizures in the PB and PHT group and 3/14 late seizures in the 'no treatment' group. Two trials (Lee 1989; North 1983) examined differences between PHT and a PCB control group. In the trial by Lee 1989 patients were treated for three days with no further follow-up, therefore only early seizures within the three days were reported. They found 2/189 seizures occurred in the PHT group compared to 9/185 in the PCB group. In the trial by North 1983, 4/140 early seizures were reported in the PHT group compared to 14/141 in the PCB group. For late seizures, 14/140 occurred in the PHT group compared to 12/141 in the control group. Nakamura 1999 conducted a head-to-head trial of ZNS versus PB. They reported 6/129 early seizures occurring within the ZNS group compared to 3/126 early seizures occurring within the PB group. For late seizures, 7/129 occurred in the ZNS group and 8/126 occurred in the PB group. Overall, only one trial (North 1983) had a significant difference between AED treatment and controls for early seizure occurrence. All other comparisons were non-significant.
Across all trials, 13/50 deaths were reported in the PHT group compared to 10/50 in the VAL group (Beenen 1999), 9/50 occurred in the CBZ (six month) group and 10/56 in the CBZ (24 month) group compared to 15/55 in the PHT (six month) group and 27/56 in the CBZ (24 month) group and 13/59 in the no treatment group (Foy 1992), 8/112 occurred in the ZNS group compared to 13/107 in the PB group (Nakamura 1999), and 20/140 occurred in the PHT group compared to 24/141 in the PCB group (North 1983). No data were collected for this outcome in two trials (Franceschetti 1990; Lee 1989).
Adverse effects
Four trials reported information on adverse effects. In the Beenen 1999 trial, 4/50 people experienced a skin reaction, 3/50 people experienced liver dysfunction, 1/50 people experienced thrombopenia and there was one case of nausea within the PHT group (N = 50). In the VAL group there were three cases of liver dysfunction, and one case of a rise in liver enzymes (N = 50). Nakamura 1999 reported two cases of somnolence and six cases of nausea in the ZNS group (N = 129), and seven cases of somnolence and two cases of nausea in the PB group (N = 126). Overall they reported 28/129 adverse effects in the ZNS group and 30/126 in the PB group. The North 1983 trial reported eight cases of rash, one case of involuntary movements, one hirsutism, one headache and one case of discomfort of the face in the PHT group (N = 140) compared to one case of rash, one dizziness and one nausea in the PCB group (N = 141). Franceschetti 1990 reported minimal data on the adverse effects, only that 3/10 people in the PHT group and 1/10 people in the PB group experienced neurological side effects. No data from the remaining trials were provided (Foy 1992; Lee 1989).
The trials included in this review were all RCTs investigating the effects of a range of AEDs given either immediately before or after a craniotomy procedure to people with no previous history of seizures or exposure to AEDs. The underlying pathologies for craniotomy surgery were mixed within the studies (e.g. tumour, abscess, meningioma) with a small percentage of patients having surgery as a result of head injuries. One study included a substantial proportion (210/374) of head-injury patients (Lee 1989). This is a major limitation of this review as the objective is to examine outcomes for patients undergoing craniotomy presenting with non-trauma pathology. We acknowledge the possibility of differences in the risk of seizure post surgery depending on the underlying pathology of the patient.
We were unable to meta-analyse any of the data and structuring a narrative summary was difficult for a number of reasons; few trials were available under each comparison examined (see Data synthesis for list of comparisons under investigation) and the interventions varied substantially with regards to duration of treatment period, dose and method of drug administration, country, methodological rigour and underlying pathologies. Reporting of outcomes differed across the trials, not all differentiated between early and late seizures and information about adverse effects of treatment was very limited. Most trials had similar inclusion and exclusion criteria. Patients undergoing supratentorial craniotomy were randomised in five of the six trials; the sixth (Nakamura 1999) did not specify the type of surgery.
For the outcome of incidence of seizures, overall the majority of trials reported no significant difference between treatment with AEDs and no treatment or treatment with AEDs and no treatment. Head-to-head drug comparisons also yielded non-statistically significant results. Only one trial (North 1983) reported any statistically significant findings, the incidence of early seizures was reduced in the AED group (PHT) compared to a PCB (P < 0.05). Overall, results from the individual trials showed no significant differences between AED treatment participants and control participants for outcomes relevant to the number of deaths and adverse effects. None of the trials examined functional outcome of patients.
The outcomes of the risk of bias assessments conducted for each trial are worthy of note. The majority of trials were rated as unclear on several of the criteria. Only two of the six trials (Beenen 1999; Foy 1992) were rated as low in bias due to the method used to generate the randomisation sequence and only one trial (Beenen 1999) used adequate methods for concealing the allocation of intervention. Most trials used adequate methods for blinding participants and outcome assessors; however, one trial was unblinded (Foy 1992) and therefore was rated as high risk of bias for this criteria. There were no protocols available for any of the trials, therefore assessing selective reporting across trials was rated as unclear. Several trials were rated as unclear as to how missing data were managed within their analysis. In most cases attrition was reported and reasons for withdrawal were described.
Authors' conclusions
Implications for practice
The results from this review show that there is not enough evidence of sufficient quality available to suggest that AED treatment can or cannot be recommended to reduce post-craniotomy seizures. There is no evidence on which to base clinical practice.
Implications for research
To evaluate the effectiveness of prophylactic treatment with AEDs in preventing seizures following cranial surgery better, more trials are needed. These trials must address the methodological weaknesses and protocol inconsistencies identified within this review including:
Data and analyses
This review has no analyses.
Appendix 1. CENTRAL search strategy
#1 MeSH descriptor Craniotomy explode all trees
#2 (craniotom*)
#3 (postcraniotom*)
#4 (supratentorial NEXT surgery)
#5 (infratentorial NEXT surgery)
#7 MeSH descriptor Seizures explode all trees
#8 (seizure*)
#11 (postoperative NEXT epilep*)
#12 (post-operative NEXT epilep*)
#13 (postoperative NEXT seizure*)
#14 (post-operative NEXT seizure*)
Appendix 2. MEDLINE search strategy
This strategy is based on the Cochrane Highly Sensitive Search Strategy for identifying randomised trials published in Lefebvre 2011.
1. exp Craniotomy/
2. craniotom$.tw.
3. postcraniotom$tw.
4. supratentorial surgery.tw.
5. infratentorial surgery.tw.
6. 1 or 2 or 3 or 4 or 5
7. exp Seizures/
8. seizure*.tw.
9. 7 or 8
10. 6 and 9
11. postoperative epilep$.tw.
12. postoperative seizure$.tw.
13. 10 or 11 or 12
14. randomized controlled trial.pt.
15. controlled clinical trial.pt.
16. randomized.ab.
17. placebo.ab.
18. clinical trials as topic.sh.
19. randomly.ab.
20. trial.ti.
21. 14 or 15 or 16 or 17 or 18 or 19 or 20
22. exp animals/ not humans.sh.
23. 21 not 22
24. 13 and 23
Contributions of authors
Jennifer Pulman and Janette Greenhalgh carried out and completed the review. Anthony Marson supervised the review. Nikola Vojvodic, Aleksandar Ristic and Dragoslav Sokic developed the protocol for this review.
Declarations of interest
None declared.
Sources of support
Internal sources
- No sources of support supplied
External sources
- National Institute for Health Research, UK.This review presents independent research commissioned by the National Institute for Health Research (NIHR). The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.
Differences between protocol and review
We were unable to make all the intended comparisons specified in the protocol due to lack of data.
Index terms
Medical Subject Headings (MeSH)
Anticonvulsants [*therapeutic use]; Carbamazepine [therapeutic use]; Craniotomy [*adverse effects]; Isoxazoles [therapeutic use]; Phenobarbital [therapeutic use]; Phenytoin [therapeutic use]; Randomized Controlled Trials as Topic; Seizures [etiology; *prevention & control]; Valproic Acid [therapeutic use]
MeSH check words
* Indicates the major publication for the study


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Paquita la del BarrioView In iTunes
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Paquita la del Barrio (born Francisca Viveros Barradas) became a celebrity after she decided to move from her native Veracruz to Mexico City in 1970. Her live performance, mainly based on feminist songs with lyrics that are aggressive toward macho attitudes, seduced followers from all around the country. Even when her first records didn't achieve a top-selling status, Paquita la del Barrio became one of the most interesting local personalities.
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BBB Accredited Business since
R & A Import Auto Parts and Repair of Flagstaff, Inc
Phone: (928) 526-2851 Fax: (928) 527-7580 2932 E Route 66, Flagstaff, AZ 86004 http://www.ethicalaz.com/raimportautopartsrepairinc
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We service Honda, Toyota, Subaru and Nissan Vehicles. Performing all Factory Recommended Services, Timing Belt replacements, Brakes, Shocks, Struts, Electrical, Check Engine Diagnostics. We are a full service shop offering both major and minor engine and transmission repair. We take pride in performing proper diagnostics and repair. We also treat all of our customers as friends or family.
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Pear And Grape Salad
29 Oct 2009
This Pear and Grape Salad tastes yummy ! Try this fruity accompaniment with cheese and lettuce for your next meal ! Your suggestions for this Pear and Grape Salad are welcome !
|Ripe dessert pears
|Cream cheese
|1 Cup (16 tbs)
|French dressing
|2 Tablespoon
|Black grapes
|1⁄2 Pound
|Crisp lettuce
1. Peel the pears, cut in half, and scoop out the core with a teaspoon.
2. Blend the cream cheese with enough French dressing to make it spreadable and coat the rounded side of each pear half.
3. Halve and seed the grapes and press them into the cheese, close together so that each pear half resembles a small bunch of grapes.
Recipe Summary
Difficulty Level:Easy
Course:Fruit Dessert
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Pear And Grape Salad Recipe


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Helping in little ways at Fair Oaks
Published 08/13/2010, Wadena Pioneer Journal
This being the year and the time helping others needs to be claiming center stage, I must mention that although Fair Oaks Lodge folks are no longer among those in the community who can help others in many ways, folks in a nursing home practice caring and helping each other in a myriad of ways never recognized. They do it all the time.Word count: 524
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