text:
These authors contributed equally to this work.
Hepatobiliary Malignancies
MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells†
Article first published online: 18 FEB 2009
DOI: 10.1002/hep.22919
Copyright © 2009 American Association for the Study of Liver Diseases
Volume 50, Issue 1, pages 113–121, July 2009
Additional Information
How to Cite
Xu, T., Zhu, Y., Xiong, Y., Ge, Y.-Y., Yun, J.-P. and Zhuang, S.-M. (2009), MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells. Hepatology, 50: 113–121. doi: 10.1002/hep.22919
- †
Potential conflict of interest: Nothing to report.
Publication History
- Issue published online: 23 JUN 2009
- Article first published online: 18 FEB 2009
- Accepted manuscript online: 18 FEB 2009 12:00AM EST
- Manuscript Accepted: 12 FEB 2009
- Manuscript Received: 25 DEC 2008
Funded by
- Ministry of Science and Technology of China. Grant Numbers: 2005CB724600, 2007AA02Z124
- National Natural Science Foundation of China. Grant Number: 30870964
- Ministry of Health of China. Grant Number: 2008ZX10002-019
Growing evidence indicates that deregulation of microRNAs (miRNAs) contributes to tumorigenesis. Down-regulation of miR-195 has been observed in various types of cancers. However, the biological function of miR-195 is still largely unknown. In this study we aimed to elucidate the pathophysiologic role of miR-195. Our results showed that miR-195 expression was significantly reduced in as high as 85.7% of hepatocellular carcinoma (HCC) tissues and in all of the five HCC cell lines examined. Moreover, introduction of miR-195 dramatically suppressed the ability of HCC and colorectal carcinoma cells to form colonies in vitro and to develop tumors in nude mice. Furthermore, ectopic expression of miR-195 blocked G1/S transition, whereas inhibition of miR-195 promoted cell cycle progression. Subsequent investigation characterized multiple G1/S transition-related molecules, including cyclin D1, CDK6, and E2F3, as direct targets of miR-195. Silencing of cyclin D1, CDK6, or E2F3 phenocopied the effect of miR-195, whereas overexpression of these proteins attenuated miR-195-induced G1 arrest. In addition, miR-195 significantly repressed the phosphorylation of Rb as well as the transactivation of downstream target genes of E2F. These results imply that miR-195 may block the G1/S transition by repressing Rb-E2F signaling through targeting multiple molecules, including cyclin D1, CDK6, and E2F3. Conclusion: Our data highlight an important role of miR-195 in cell cycle control and in the molecular etiology of HCC, and implicate the potential application of miR-195 in cancer therapy. (HEPATOLOGY 2009.)
It has been well demonstrated that aberration of protein-coding genes plays a crucial role in the development of human cancers. Recently, growing evidence has indicated that deregulation in noncoding genes, particularly microRNAs (miRNAs), also contributes to tumorigenesis.1, 2
miRNAs belong to a class of short, highly conserved noncoding RNAs known to suppress the expression of protein-coding genes through imperfect base-pairing with the 3′-untranslated region (3′ UTR) of target messenger (m)RNA.3 It is predicted that one-third of protein-coding genes in humans are regulated by miRNAs.4 Indeed, miRNAs have been implicated in the control of various biological processes, such as development,5 cell proliferation, apoptosis,6 and differentiation.7 Additionally, more than half of the annotated human miRNA genes are located in chromosomal regions displaying frequent amplification, deletion, or translocation in human cancers.8 Furthermore, deregulation of miRNAs has been observed in different types of human malignancies1, 9–14 and is also associated with the clinical outcome of cancer patients.11, 12, 15 Accumulating evidence suggests that miRNAs may act as oncogenes or tumor suppressor genes.2
Using miRNA microarray, we and others have previously shown down-regulation of the miR-15/16/195 family members in hepatocellular carcinoma (HCC) as well as other human neoplasms.9, 12–14 It was recently reported that ectopic expression of this family's members results in down-regulation of cell-cycle-related transcripts, including CDK6, CCNE1, Cdc25A, and Chk1, and causes an accumulation of G1 cells.16 However, the signaling pathways regulated by the miR-15/16/195 family and the exact role of this family in tumorigenesis are still largely unknown.
It has been demonstrated that members of an miRNA family usually play overlapping, if not identical, roles,17–21 because they share common binding sequences for target recognition. Therefore, miR-195, which, among the miR-15/16/195 family members, displayed the most significantly reduced expression in HCC in our previous study,9 was chosen as representative to clarify the pathophysiologic role and the relevance of the miR-15/16/195 family to tumor biology. We showed that down-regulation of miR-195 occurred very frequently in HCC tissues and cell lines. Furthermore, ectopic expression of miR-195 dramatically suppressed HCC cells to form colonies in vitro and to develop tumors in nude mice. Moreover, both gain- and loss-of-function studies revealed that miR-195 could block the G1/S transition. Multiple G1/S transition-related molecules, including cyclin D1, CDK6, and E2F3, were further characterized as direct functional targets of miR-195. These findings suggest an important role of miR-195 in cell cycle control and in the molecular etiology of HCC, with implications for cancer therapy.
Materials and Methods
Cell Lines and Tissue Specimens.
Detailed information about the HCC cell lines and tissue specimens is described in the Supporting Materials and Methods. The relevant characteristics of the studied subjects are shown in Supporting Table 1.
RNA Oligoribonucleotides.
miRNA duplexes corresponding to miR-195, miR-16, and miR-497 were designed as described.22 The small interfering (si)RNAs targeting human cyclin D1 (GenBank Access. No. NM_053056), CDK6 (NM_001259), E2F3 (NM_001949), and c-Myc (NM_002467) transcripts were designated siCCND1, siCDK6, siE2F3, and siMyc, respectively. The negative control RNA duplex (denoted NC), designed based on cel-miR-67, has been demonstrated to have minimal sequence identity with human miRNAs (Dharmacon, Lafayette, CO). For the in vivo tumorigenicity assay, all pyrimidine nucleotides in the NC or miR-195 duplex were replaced by their 2′-O-methyl analogs to improve RNA stability. The anti-miR-195, with a sequence complementary to the mature miR-195, was a 2′-O-methyl-modified oligoribonucleotide. The anti-miR-C was used as a negative control for anti-miR-195. All RNA oligoribonucleotides (Supporting Table 2) were purchased from Genepharma (Shanghai, China).
Semiquantitative Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR), Northern and Western Blot.
Primers used for semiquantitative RT-PCR and probes used for Northern blot analysis are listed in Supporting Table 2.
Northern blot was conducted as reported.23 We first optimized 55°C as the proper hybridization temperature to distinguish miR-195 from miR-16, which shares the highest sequence similarity with miR-195 (Supporting Fig. 1).
The antibodies used for Western blot are described in the Supporting Materials and Methods.
RNA and protein bands were quantified using GeneTools software (version 3.03; SynGene, Cambridge, UK).
Cell Transfection.
Reverse transfection of RNA duplex was performed using Lipofectamine-RNAiMAX (Invitrogen, Carlsbad, CA). All RNA transfections were performed at a final concentration of 50 nM unless otherwise indicated. The transfection efficiency, determined by a fluorescein amidite (FAM)-conjugated siRNA (nonhomologous to any human genome sequences) and fluorescence-activated cell sorting (FACS) analysis, was about 79% for MHCC-97L and 71% for HCT-116 cells (data not shown). Transfection of plasmid DNA was performed using calcium phosphate precipitation for 293T cells and Lipofectamine 2000 (Invitrogen) for other cell lines.
Analysis of Clonogenicity In Vitro and Tumorigenicity in Nude Mice.
Analyses for clonogenicity in low cell density, anchorage-independent cell growth, and tumorigenicity were performed as described in the Supporting Materials and Methods.
Analysis of Cell Proliferation.
Cell Cycle Analysis.
Except for rescue experiments, all cell cycle analyses were performed using a detergent-containing hypotonic solution (Krishan's reagent) and FACS (Becton Dickinson, San Jose, CA), as reported.24 Nuclear debris and overlapping nuclei were gated out unless stated otherwise.
For rescue assay, the DNA content of living cells was measured by FACS using Hoechst 33342, as described in the Supporting Materials and Methods.
Vector Construction and Luciferase Reporter Assay.
The expression vectors pc3-miR-195, pc3-gab-CCND1, pc3-gab-CDK6, and pc3-gab-E2F3 were generated as described in the Supporting Materials and Methods. To create a luciferase reporter construct, 3′ UTR fragment containing putative binding sites for miR-195 was inserted downstream of the stop codon of firefly luciferase in pGL3cm as described.9 Mutant 3′ UTR, which carried a mutated sequence in the complementary site for the seed region of miR-195, was generated using the fusion PCR method.
293T cells grown in a 48-well plate were cotransfected with 200 ng of either pcDNA3.0 or pc3-miR-195, 10 ng of firefly luciferase reporter comprising wildtype or mutant 3′ UTR of target gene, and 2 ng of pRL-TK (Promega, Madison, WI). The luciferase assay was performed as reported.9
Paraffin-embedded, formalin-fixed tissues were immunostained for E2F3, cyclin D1, and CDK6 proteins, as described in the Supporting Materials and Methods.
Statistical Analysis.
Data are expressed as the mean ± SEM from at least three separate experiments. Unless otherwise noted, the differences between groups were analyzed using Student's t test when only two groups, or assessed by one-way analysis of variance (ANOVA) when more than two groups were compared. All tests performed were two-sided. Differences were considered statistically significant at P < 0.05.
Expression of miR-195 Is Frequently Reduced in HCC Tissues.
In a previous microarray analysis, we found that miR-195 was down-regulated in HCC tissues compared with normal liver tissues.9 To further confirm and extend this finding, we analyzed the expression of miR-195 in 14 paired HCC and adjacent noncancerous liver tissues by Northern blot. Compared with their noncancerous counterparts, significant down-regulation of miR-195 was observed in 85.7% (12/14) of HCC samples (Fig. 1). Furthermore, 9 out of 14 (64.3%) HCC displayed more than 50% reduction in the miR-195 level. Down-regulation of miR-195 was also found in all five HCC cell lines examined (Supporting Fig. 2). These results suggest that reduced miR-195 expression is a frequent event in human HCC and may be involved in hepatocarcinogenesis.
miR-195 Suppresses Clonogenicity In Vitro and Tumor Growth In Vivo.
To examine the potential role of miR-195 in tumorigenesis, we first evaluated the effect of miR-195 on the growth and clonogenicity of cancer cells. Two HCC cell lines (MHCC-97L, Hep3B) were transfected with NC or miR-195 duplex, and then allowed to grow at very low density. Notably, miR-195–transfected cells displayed obviously fewer and smaller colonies compared with NC-transfectants (Fig. 2A), suggesting a growth-inhibitory role of miR-195. To test whether this observation could be generalized to other solid tumor cells, miR-195 was transfected into colorectal carcinoma (HCT-116) and osteosarcoma (U-2OS) cells. Interestingly, both of them also exhibited dramatically decreased colonies after miR-195 expression (Fig. 2A), indicating a ubiquitous role of miR-195 in cell growth. This growth-inhibitory effect of miR-195 was also confirmed by Alamar Blue assay (Supporting Fig. 3).
We further analyzed the effect of miR-195 on anchorage-independent cell growth and found that miR-195 dramatically repressed the capability of cancer cells to grow in soft agar, as shown by a significant reduction both in the size and the number of colonies (Supporting Fig. 4). The above findings were further confirmed by an in vivo xenograft model. NC- and miR-195-tranfected MHCC-97L cells were injected subcutaneously into either posterior flank of the same nude mice. The mice were followed for observation of xenograft growth for 4 weeks. We found that introduction of miR-195 into MHCC-97L cells led to a significant reduction in both the frequency of tumor formation (NC versus miR-195-transfectants, 5/5 versus 3/5) and the size of tumor volume (Fig. 2B,C, upper panel). This suppression on tumorigenicity was also reproducible in HCT-116 xenografts. Tumors appeared in the sites injected with NC-transfected HCT-116 in 56% (5/9) of mice, and became palpable 12-22 days after inoculation and grew to 146∼1,386 mm3 at the end of observation (4 weeks). In sharp contrast, no tumors were observed in the flanks injected with miR-195-transfectants through the observation period (Fig. 2B,C, lower panel).
Collectively, both in vitro and in vivo studies suggest that miR-195 significantly inhibits tumorigenicity.
Ectopic Expression of miR-195 Blocks G1/S Transition.
To explore the mechanisms underlying miR-195-suppressed tumor growth, predicted target genes of miR-195 were retrieved from the TargetScan database and their functions were categorized based on the Protein Lounge Pathway Database (http://www.proteinlounge.com). The enrichment of predicted targets in defined biological pathways was then evaluated by a chi-square test. Interestingly, we found a significant enrichment of cell cycle-related genes among the predicted targets (4.2-fold overrepresentation, P < 10−4; Supporting Table 3). To confirm this finding, target prediction for miR-195 was further performed using another algorithm, miRanda,25 with minimal score and free energy set as 90 and −17 cal/mol, respectively. A similar trend of enrichment in cell cycle-related transcripts was also observed (5.1-fold overrepresentation, P < 10−7; Supporting Table 3).
Such an enrichment prompted us to investigate the impact of miR-195 on cell cycle progression. The results revealed that enforced expression of miR-195 caused marked accumulation of a G1 population in different cell lines, whereas little effect on apoptosis was observed as evaluated by sub-G1 peak (Supporting Fig. 5). To further confirm this finding, we synchronized the transfected cells with nocodazole, which depolymerizes the microtubules and blocks cell cycle at G2/M-phase. Thirty-two hours after transfection, cells were incubated with nocodazole for an additional 16 hours before FACS analysis. Consistently, miR-195-transfected cells showed a substantial rise in the G1 population (Fig. 3A and Supporting Fig. 6A). Similar results were obtained when cells were transfected with miR-16 or miR-497 (Supporting Fig. 7), two other members of the miR-15/16/195 family, in agreement with the general concept of functional redundancy among paralogous family members.
Next, a serum starvation-stimulation assay was employed to precisely clarify whether miR-195-induced accumulation of G1-cells resulted from a blocked G1/S transition. MHCC-97L, HCT-116, and U-2OS cells were transfected with NC or miR-195 for 24 hours, followed by synchronization with serum deprivation for 48 hours and then stimulation to enter S-phase by serum readdition. Interestingly, when a large proportion of NC-transfected cells already entered S-phase, most of the miR-195-transfectants still stayed in G1-phase after serum stimulation (Fig. 3B and Supporting Fig. 6B), suggesting that miR-195 blocks the G1/S transition. This contention was further supported by BrdU incorporation assay (Supporting Fig. 6C).
miR-195 Directly Inhibits the Expression of Cyclin D1, CDK6, and E2F3 through Their 3′ UTRs.
To unravel the mechanism by which miR-195 restrains the G1/S transition, we searched for positive regulators of a G1/S transition among the predicted targets of miR-195. Cyclin D1, CDK6, and E2F3 stood out as attractive candidates, because they are crucial components that initiate the inactivation of the Rb pathway and in turn the G1/S transition. We were able to map a single putative miR-195-binding site in the 3′ UTR of E2F3 and two sites in each 3′ UTR of cyclin D1 and CDK6 (Supporting Fig. 8). To validate whether these genes were direct targets of miR-195, a dual-luciferase reporter system was first employed. Firefly luciferase reporter containing wildtype or mutant 3′ UTR of the target gene was cotransfected with Renilla luciferase reporter and either pcDNA3.0 or pc3-miR-195. Coexpression of pc3-miR-195 significantly suppressed firefly luciferase activity of the reporter with wildtype 3′ UTR but not that of the mutant reporter (Fig. 4A), indicating that miR-195 may suppress gene expression through its binding sequences at the 3′ UTR of target genes.
The effect of miR-195 on endogenous expressions of cyclin D1, CDK6, and E2F3 was subsequently examined. Transfection of miR-195 induced an obvious decrease in all three proteins in both MHCC-97L and U-2OS cells, and in CDK6 and E2F3 in HCT-116. In addition, miR-195 overexpression caused a reduced mRNA level in CDK6 but not in cyclin D1 and E2F3 (Fig. 4B and Supporting Fig. 6D).
We next examined the correlation between the decreased expression of miR-195 and the overexpression of E2F3, cyclin D1, and CDK6, the targets of miR-195, in HCC tissues. E2F3, cyclin D1, and CDK6 were analyzed by immunohistochemistry in the same set of specimens shown in Fig. 1. Notably, E2F3 was highly expressed in 71.4% (10/14) of HCC tissues, but undetectable in all of the corresponding noncancerous samples (Supporting Table 1 and Fig. 4C). Importantly, 10 out of 12 HCC displaying a striking decrease in the miR-195 level revealed enhanced expression of E2F3, whereas those two HCC without an obvious reduction of miR-195 expression showed undetectable E2F3 staining (cases 729 and 727; Supporting Table 1; Figs. 1, 4C). Consistently, statistical analysis revealed that the relative miR-195 level (as measured by the tumor/nontumor [T/N] ratio) was significantly lower in HCC with E2F3 overexpression (T/N = 0.37 ± 0.06) than those without E2F3 staining (T/N = 0.74 ± 0.20; P < 0.05 by t test). These data suggest that HCC tissues with lower miR-195 levels predispose to E2F3 overexpression.
We also found that cyclin D1 and CDK6 were overexpressed in 21.4% (3/14) and 85.7% (12/14) of samples, respectively (Supporting Table 1). Furthermore, a trend of correlation between the decreased miR-195 level and the cyclin D1 or CDK6 overexpression was also observed. The relative miR-195 level in HCC with cyclin D1 or CDK6 overexpression was about 2-fold lower than those without enhanced expression of cyclin D1 or CDK6 (T/N = 0.24 ± 0.01 versus 0.53 ± 0.09 for cyclin D1; T/N = 0.41 ± 0.06 versus 0.85 ± 0.38 for CDK6), although the correlation did not reach statistical significance (P = 0.14 for cyclin D1; P = 0.05 for CDK6 by t test), which might be due to the limited sample size after stratification based on the expression status of cyclin D1 or CDK6 (n ≤ 3 in the minor group).
Taken together, these data imply that miR-195 may attenuate the expression of cyclin D1, CDK6, and E2F3 by directly targeting their 3′ UTRs.
Cyclin D1, CDK6, and E2F3 Are Involved in miR-195–Regulated G1/S Transition.
To explore the role of cyclin D1, CDK6, and E2F3 in miR-195-regulated G1/S transition, we examined whether knockdown of these genes may mimic the effect of miR-195 overexpression. Cells were transfected with siRNA duplex targeting either cyclin D1, CDK6, or E2F3, which resulted in a significant reduction in both mRNA and protein levels of the respective genes (Fig. 4B and Supporting Fig. 6D). Markedly, except for knockdown of cyclin D1 in MHCC-97L, silencing of either target genes led to substantial arrest of cell cycle in G1-phase (Fig. 3A and Supporting Fig. 6A), phenocopying the outcome of miR-195 expression. Interestingly, compared with miR-195 overexpression, inhibition of cyclin D1, CDK6, or E2F3 alone caused a less extent of G1-arrest, implying that miR-195 may repress the G1/S transition through synergistically targeting multiple targets. We subsequently examined whether cyclin D1, CDK6, and E2F3 could counteract the effect of miR-195. Notably, overexpression of these proteins partially rescued miR-195-induced G1-arrest in MHCC-97L and U-2OS cells (Fig. 5A and data not shown).
Cyclin D1 and CDK6 are crucial molecules that initiate the phosphorylation of Rb, which results in the release of E2F and in turn the transactivation of genes required for S-phase entry. We therefore explored whether miR-195 could attenuate these events. Interestingly, ectopic delivery of miR-195 caused a prominent decrease of phosphorylated-Rb protein (ppRb) (Fig. 5B and Supporting Fig. 6E). Furthermore, the mRNA levels of downstream targets of E2F, including Mcm3, DHFR, TK1, Cdc6, cyclin E1, and cyclin A2 genes, were significantly lower in miR-195-transfected cells than in NC-transfectants (Fig. 5C and Supporting Fig. 6F).
These data suggest that miR-195 may inhibit the G1/S transition by suppressing multiple targets including cyclin D1, CDK6, and E2F3, and in turn attenuating the transcription of S-phase genes.
Inhibition of miR-195 Promotes G1/S Transition.
To confirm the results from the gain-of-function study, we performed loss-of-function analysis using anti-miR-195, an inhibitor of miR-195. Transfection of anti-miR-195 dramatically counteracted the G1-arrest triggered by exogenous miR-195 (Fig. 6A). The outcome of antagonism on endogenous miR-195 was further investigated. Compared with anti-miR-C transfection, anti-miR-195 induced a statistically significant decrease of G1-cells (50.4% versus 60.3%, P = 0.003, Fig. 6B), although the extent of antagonism was not as evident as that on exogenous miR-195, which might be explained by the low basal level of miR-195 in cancer cells (Supporting Fig. 2).
It has been shown that c-Myc-silencing can induce G1-arrest by reducing the expression of cell cycle-related molecules,26–28 including cyclin D1 and E2F3, the targets of miR-195 identified in this study. We therefore attempted to further confirm the effect of anti-miR-195 on endogenous miR-195 using a c-Myc-knockdown model, in which the increased G1 population and the reduced expression of cyclin D1 and E2F3 may make the antagonism of anti-miR-195 more pronounced. HCT-116, a cell line with a high level of endogenous c-Myc, was transfected with siMyc, which significantly repressed c-Myc expression (Supporting Fig. 9). As expected, compared with NC-transfected cells, siMyc-transfectants displayed an increased proportion of G1-cells (Fig. 6C) and a reduced level of cyclin D1, CDK6, E2F3, and ppRb (Fig. 6D). Surprisingly, transfection of anti-miR-195 not only significantly enhanced the levels of cyclin D1, CDK6, E2F3, and ppRb, but also dramatically relieved the G1-arrest in c-Myc-knockdown cells (Fig. 6C,D).
These findings, together with the data from gain-of-function experiments, point to a role of miR-195 in cell cycle regulation.
It is well demonstrated that a defect in cell cycle control is an essential step during carcinogenesis. Therefore, it is reasonable to expect that deregulation of cell cycle-related miRNAs may facilitate tumorigenesis. In this study we showed that miR-195 was frequently down-regulated in both HCC tissues and cell lines. Ectopic expression of miR-195 suppressed HCC and colorectal carcinoma cells to form colonies in vitro and to develop tumors in vivo. Moreover, gain- and loss-of-function studies revealed that miR-195 could block the G1/S transition by repressing the Rb-E2F signaling through multiple targets, including cyclin D1, CDK6, and E2F3. We propose that reduced expression of miR-195 may disrupt cell cycle control, in turn promote cell proliferation, and consequently facilitate the development of cancers like HCC.
The underlying mechanism responsible for decreased expression of miR-195 in HCC is still unknown. Notably, miR-195 is located at chromosome 17p13.1, a region that is frequently deleted in human cancers, especially HCC. In addition, we were able to computationally map a CpG-enriched region upstream of the miR-195 gene. Therefore, allelic loss and promoter hypermethylation might partially account for the reduced miR-195 expression in cancers.
The miR-15/16/195 family has been shown to promote apoptosis in leukemia cells,29 whereas this effect has not been observed in the cell models used in the present study and previous reports.16, 30 This discrepancy is likely due to the difference in cell context, and suggests that altered expression of this miRNA family may have diverse effects in tumor cells. Nevertheless, the similar effects of miR-195 in cell lines derived from different solid cancers implicate its fundamental role in cell cycle regulation and tumorigenesis.
Intriguingly, the promoting effect of anti-miR-195 on cell cycle progression was exaggerated after c-Myc-knockdown (Fig. 6B,C). We speculate that c-Myc-silencing may provide a more proper condition for examining the effect of miR-195 inhibition. For example, the anti-miR-195-induced increase in the expression of miR-195 targets may become more evident when these targets are expressed at low levels after c-Myc-knockdown. Besides, a recent study showed that c-Myc could repress the transcription of miR-195/497 and miR-15a/16 polycistrons in lymphoma cells.31 However, we could not find an obvious elevation in either miR-195 and miR-16 levels after c-Myc-silencing (Supporting Fig. 10).
Although miR-195 effectively suppressed cyclin D1 expression in diverse cell lines examined in our and other studies,30 we noticed that none of miR-195, miR-16, and miR-497 were able to reduce the level of cyclin D1 in HCT-116 cells (Fig. 4B and Supporting Fig. 11). It is becoming increasingly clear that miRNA-mediated suppression of gene expression is subjected to intense regulation. For example, the repression of CAT-1 by miR-122 in HCC cells is relieved under stress conditions by binding of HuR to the 3′ UTR of CAT-1.32 Furthermore, it has been shown that Dnd1 protein can inhibit the access of miRNA to target mRNA, and therefore block the regulation function of miRNA on gene expression.33 It will be interesting to characterize how the interaction between miR-195 and the mRNA of cyclin D1 is controlled in HCT-116.
While this article was in preparation, Liu et al.30 reported that the miR-15/16/195 family directly targeted cyclin D1 and CDK6, and forced expression of either miR-16 or miR-195 increased G1-cell proportion in a lung cancer cell line. In the present study, we confirmed and extended their findings in other cell models. We provided evidence to support the potential tumor suppressive activity of miR-195, further defined miR-195 as an inhibitor of G1/S transition, and characterized E2F3, a transcription factor downstream of Rb, as another target of miR-195. It is well known that the Rb pathway acts as a master checkpoint in cell cycle progression. The integration of miR-195 in the Rb-E2F pathway further demonstrates that cell cycle is regulated by elaborate mechanisms, to prevent uncontrolled cell proliferation. Our finding that miR-195 targets molecules both upstream (e.g., cyclin D1, CDK6) and downstream (e.g., E2F3) of Rb, an archetypal tumor suppressor, provides new insight into a fail-safe mechanism, which should guard the cells from runaway proliferation in the case of aberrant Rb-inactivation, which is frequently found in human HCC.34
In summary, we investigated the potential role of miR-195 in cell cycle control and in tumorigenesis. Our data suggest an important role of miR-195 in the molecular etiology of cancer and implicate its potential application in cancer therapy.
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Supporting Information
Additional Supporting Information may be found in the online version of this article.
|Supporting Materials and Methods
|Supplementary Table 1. The Characteristics of Patients and Expression Status of miR-195 and Its Target Genes
|Supporting Figure 1. Specific detection of miR-195 by Northern blot. (A) Optimal hybridization temperature for specific detection of miR-195. Five pmols of synthetic RNA duplexes corresponding to miR-195 (lane 1) and miR-16 (lane 2) were separated on 15% denaturing polyacrylamide gel. Hybridizations were performed at indicated temperatures. The probe sequence was complementary to mature miR-195. (B) Sequence alignment of mature miRNA sequences of the miR-15/16/195 family members. Shading letters indicate the nucleotides same as mature miR-195.
|Supporting Figure 2. Expression level of miR-195 in different cell lines and normal human liver tissue. The same membrane was hybridized sequentially with miR-195 and U6 probe, as indicated on the left. HCC, hepatocellular carcinoma cell lines; U6, control for RNA loading.
|Supporting Figure 3. miR-195 expression reduces the number of viable cells. Cells were first transfected with NC or miR-195 duplex in a 24-well plate for 24 h, and then re-plated into 96-well plate at about 10% confluence, followed by Alamar Blue assay at every 24 h for 5 days, indicated as Day 1 to 5. *, P < 0.05; ***, P < 0.001, compared with NC-transfectants.
|Supporting Figure 4. miR-195 suppresses anchorage-independent cell growth. Representative pictures (A) and quantitative plots (B) are shown. Colonies larger than 150 μm in diameter were counted under 30 separate microscopic fields at 150 × magnification. ***, P < 0.001, compared with NC-transfectants.
|Supporting Figure 5. Ectopic expression of miR-195 induces accumulation of G1-cells in hepatocellular carcinoma (HCC), colorectal carcinoma (CRC) and osteosarcoma cells. For measurement of apoptotic cells, nuclear debris detected as sub-G1 peak were included. The percentage of G1 population is indicated within each histogram.
|Supporting Figure 6. Rb-E2F signaling is involved in miR-195-regulated G1/S transition in U-2OS cells. (A) Overexpression of miR-195 and knockdown of either cyclin D1, CDK6 or E2F3 increase the proportion of G1-cells. (B-C) miR-195 expression delays S-phase entry upon serum stimulation as assessed by FASC analysis (B) and BrdU incorporation assay (C). **, P < 0.01; ***, P < 0.001, compared with NC-transfectants. (D) Suppressed expression of endogenous cyclin D1, CDK6 and E2F3 by miR-195 or siRNA. Forty-eight hours after transfection with indicated RNA duplex, endogenous mRNA and protein levels of cyclin D1, CDK6 and E2F3 were analyzed by RT-PCR and Western Blot, respectively. (E) Phosphorylation of Rb protein is inhibited by miR-195. pRb, total Rb protein; ppRb, phosphorylated pRb. (F) Transactivation of E2F3 target genes is impeded by miR-195. The time point when serum was re-added was set as 0 h. Gene expression was examined by RT-PCR. β-actin or hPRT was used as internal control.
|Supporting Figure 7. Effect of miR-16 and miR-497 overexpression on the G1-population. HCT-116 cells were transfected with indicated miRNA duplex. Nocodazole (40 ng/ml) was added 32 h after transfection and cells were cultured for additional 16 h, followed by FASC analysis. ***, P < 0.001, compared with NC-transfectants.
|Supporting Figure 8. Putative miR-195 binding sites in the 3'UTR of respective genes. Mutation was generated in the complementary site (underlined) for the seed region of miR-195, as indicated.
|Supporting Figure 9. Examination of c-Myc expression by RT-PCR and Western blotting. (A) Expression of endogenous c-Myc in U-2OS, MHCC-97L and HCT-116 cells. (B) Knockdown of c-Myc expression in HCT-116 cells by 5 nM of siMyc. β-actin was used as internal control.
|Supporting Figure 10. Expression level of miR-195 and miR-16 after c-Myc knockdown. Levels of miR-195 and miR-16 were analyzed by real-time RT-PCR assay (PN 4373105 and PN 4373121, ABI, Foster City, CA) at indicated time points after transfection with siMyc or NC duplex in HCT-116 cells. RNU49 was used as internal control (PN 4373376, ABI). Fold change of expression indicates miRNA expression in si-Myc-transfectants relative to that of the matched NC-transfectants.
|Supporting Figure 11. Effect of miR-195, miR-16 or miR-497 overexpression on endogenous cyclin D1 expression of HCT-116 cells. Forty-eight hours after transfection with indicated RNA duplex, endogenous protein levels of cyclin D1 was assayed by Western Blot. β-actin was used as internal control.
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


text:
Pro Tour players like Jason Day and veteran short-game threat, Dave Stockton, are hyping up this new putter from TaylorMade's proven Ghost Series. Called the Spider, its design is certainly intriguing. It's easier to align & aim and also offers adjustable weight technology letting you custom-tune the feel of the club.
There's only one down side to waking up at noon. To eat breakfast or lunch? The Denver burger splits the difference. It's a traditional burger topped with Wisconsin cheddar, and topped again, with a Swiss-cheese demi-omelette. Yeah … a burger with an omelet on top. And that's not all, click below to read the rest of the recipe.
Due to lack of evidence, actor Richard Gere was never prosecuted for "gerbiling", but he was taken to the cleaners in the court of public opinion. Regardless, he was able to deftly play the role of a powerful, fame-hungry lawyer in this 1996 legal thriller. Starring Ed Norton as the killer on trial, Gere gets tangled up in big city politics as the murder of the city's archbishop unfolds. Can Gere get Norton off? You'll have to watch and find out.
Beer is pretty much a go anywhere, everywhere, every day. This we know. But now, the beer aficionados at Thirsty Swagman have developed a beer tour making even space travel fun. They fly you 62 miles up and then crack beers on the spaceship. That's right, sign up for their incredible $95K tour and you can drink a brew in Zero Gravity.
With ten years of Le Mans victories under their belt, Audi is launching a commemorative model of their bad-ass R8. Packing the same heat as their 430-horse V8, this upgrade is all cosmetic. Titanium-effect paint, 19-inch Double-Y spoke wheels with red brake calipers stuck in behind them, plus a whole array of interior upgrades, make this little race car all the more racy.
For years, V-Moda's V80 headphone has been a top choice for DJs and fans of studio-quality, over-the-ear headphones. Now, they're releasing a collab model with the hit cable-TV series True Blood. Unlike the sexed-up, bloodthirsty stars of this show, these headphones don't suck.
Don't get your tool confused with the junk and other stuff cluttering your workbench. If you want to get the job done fast & easy, just hang your tool out where it's easy to find. These ½" magnetic dots stick to the wall and they're strong enough to hold a half-pound tool. If you want to hang up something bigger, add more dots.
Nobody wants to waste a perfectly good summer weekend half-assing their home improvement projects. This boss 18-volt cordless hammer drill from Bosch is lightweight, but heavy-duty. You'll have no more half-sunk screws and almost-drilled holes. This thing digs in deep & gets 'er done.
Depending on how strong you make your morning Joe, this hand grenade coffee mug may in fact cause an explosion. And remember, nobody in your office wants to be fragged in the employee bathroom. Please drink responsibly.
All the caveman needed was a campfire and a sharp stick to cook his kill. But, an evolved gentleman like yourself might appreciate a bit more know-how and craft. This book is a celebration of the art of Old World butchery; full of tips and info on the hows & whys of proper meat preparation. Filled with insights from chefs and butchers, this book is a comprehensive guide to being a good little carnivore.

subset: commoncrawl


text:
A Range-Only Tracking Algorithm for Wireless Sensor Networks
Free registration required
Executive Summary
Over the last few years, target tracking in wireless sensor networks has become a topic of particular interest. This paper presents a tracking system intended for deployment in distributed Wireless Sensor Networks. The approach is inspired from the concept of deploying sensor nodes in an ad hoc manner and based on the aggregate amount of information they provide, perform tracking of mobile objects. Static sensor nodes, with known locations, act as anchor nodes providing range-only measurements at each time step. The proposed system is modeled using a nonlinear state-space model which reflects a real world tracking scenario. The batch of range measurements made available at each sampling step, is used to estimate the target's desired kinematic properties.
- Format: PDF
- Size: 262.16 KB


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This was my first purchase from MJM Magic where I found the service excellent. I needed guidance with one of the products and Jeff went the extra...Read More ->
First time ordering with MJM. It was GREAT.
I blotched my payment method and I requested to change my order. MJM responded back within a few hours...Read More ->


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St Petersburg Mobile Home Rental Photos and Description
2 bedroom/ 1 1/2 bath with screened in Porch
This place brings you back to OLD Florida nestled in the quiet gulf coast community of sunny St. Petersburg. Our mobile home features 2 bedrooms, 1-1/2 baths, eat in kitchen, stove, microwave, refrigerator, central heat and air. Dishes, and linens provided. All you need to do is buy groceries. This older-style, charming mobile home is perfect for snowbirds, retireees, or anyone looking to get away from the colder northern regions. If you are looking for the conveniences of home, in a charming, safe and quiet location, this place is for you. Conveniently located and only a short drive to shopping, restaurants and just a 15 minute drive to the beautiful gulf beaches. Inquire now. (Ask about our larger rental right next door if you have additional couples coming -- Stay next to your friends/family! Book together!)
- Suitability:
- pets not allowed
About Sharon or Todd Gisclair
Sharon was born in upstate New York and has lived in Florida for 20 years with her husband Todd & dog and cat. After her parent's purchased a mobile home in Florida for their retirement years to escape the winters, she too bought a couple to rent out in the same community and enjoys this side hobby greeting both returning tenants and new guests.
Why Sharon or Todd Gisclair chose St Petersburg
Conveniences of home, yard, privacy, quiet and peaceful area. 15 min. drive to gulf beaches.
What makes this mobile home unique
Price is fully inclusive including water, electric, gas, phone, cable and internet. All you need to do is buy groceries.


text:
Winnipeg Free Press - PRINT EDITION
Snooping in AP phone records bad for justice
When the government obstructs the ability of a news organization to do its job, the most important victims are not journalists but readers. That's why long-standing guidelines set clear limits on how U.S. Justice Department prosecutors are expected to behave when interacting with the news media. "In recognition of the importance of freedom of the press to a free and democratic society," the government's own rules say, subpoenas for reporters' records or notes must be personally authorized by the attorney general, may be issued only after other sources are exhausted and are expected to be limited in scope to avoid gratuitous invasion into news gathering.
Yet no such restraint or respect for limits was evident in the government's wholesale sweeping up of phone records of the Associated Press, which was disclosed this week.
Related Items
The records appear to have been seized as part of an ongoing investigation into who leaked details to the AP in 2012 about a foiled terrorist plot in Yemen. On Monday, the AP said the government had gathered information from at least 20 phone lines, including the personal cellphones of some reporters and an editor, as well as the main switchboard of the AP's Washington bureau. Although the contents of the calls were apparently not recorded, ingoing and outgoing numbers were included in the information provided by the phone companies.
It's easy to see why investigators might want that information. If they can connect phone numbers belonging to government officials with reporters who put together the story, prosecutors may be able to figure out who leaked the information.
The AP described the seizure of the records as an "unprecedented intrusion." Sadly, that's not true. In fact, the Obama administration has been overzealous and irresponsible in its pursuit of leakers, aggressively striking back when it believes government officials may have illegally shared information with reporters. James Risen of the New York Times, for instance, had personal phone and bank records seized by the government as it attempted to uncover his sources. And authorities have launched five other such cases in recent years, more than any previous administration.
These inquiries are often protracted and fruitless, but their real cost lies elsewhere. The reason for limiting Justice Department searches of journalists and news organizations is that the nation benefits from aggressive reporting that holds government to account. Retaliatory leak investigations and unjustified, open-ended searches such as that performed on the AP discourage that reporting and cow sources into submission. Such searches may simplify the work of investigators, but they thwart the larger purpose of free debate, a grave sacrifice indeed. What's good for Justice may be bad for justice.
Republished from the Winnipeg Free Press print edition May 16, 2013 A13
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Europe Publications
72. Privatization in Brcko District: Why It Is Different and Why It WorksJul 07, 2011
April 2004 - The Brcko District of Bosnia and Herzegovina is small, with less than 100,000 people. In 2001, about 30 public companies were selected for privatization. At the outset, there were good reasons to ask whether the District would have any success in privatizing them. Many of the public companies had been shut down for up to ten years, while the rest were operating at a small fraction of their pre-1991 output. more
212. Geopolitical and Geostrategic Aspects of War in the Former Yugoslavia: 1991-2000Jul 07, 2011
October 2000 - The war in the former Yugoslavia was not a civil war as often asserted, but a war of aggression by the Serbian regime in Belgrade, led Slobodan Milosevic, with the aim of creating a "Greater Serbian" state. This Greater Serbia was to encompass all the Serbs that lived in the former Socialist Federal Republic of Yugoslavia (SFRY). Belgrade's regime provided strong political and propaganda support to the Serbs in Croatia and Bosnia to declare their regions autonomous. In both republics, the former Serbian-dominated Yugoslav People's Army (YPA) was used to arm the Serbian rebels and protect their self-declared autonomous areas. The YPA's attack against Slovenia in June 1991, and subsequent aggression against Croatia in July 1991, as well as against Bosnia in April 1992, were not spontaneous and improvised acts, but a part of a series of plans drawn up by the YPA's General Staff in late 1980s. more
275. East Central Europe between Paris and WashingtonJul 07, 2011
For some, Poland's emergence as a leading partner in the US-led campaign to rebuild Iraq came as a surprise. After all, so much effort has been spent on reforms, concessions and negotiations in preparation for EU accession, that it seemed to many that Poland, the Baltic States, Romania, Bulgaria and the Czech Republic were antagonizing precisely those countries they had been courting for years by supporting the US-led coalition. In response to the confusion, I would like to offer a historical-cultural explanation for those puzzled by this new world order. more
357. Romanian Parliamentary Elections: New Alliances and ChallengesJul 07, 2011
December 2008 - In December 2008, a friend in Bucharest sent me a message quoting a recent statement by an influential political commentator from the Romanian media. This columnist reminds me of the former spokesman for the Polish military junta in the 1980s, who has since become a very successful capitalist: Jerzy Urban. Urban is the editor of the weekly magazine Nie, which irreverently makes fun of everybody. In my mind, Urban is no hero, but is a former Communist Party lackey who turned into the transition's profiteering buffoon. So, I am referring here to somebody who is the equivalent of Urban in Romania, and his name is Ion Cristoiu. more
55. National Identity and Cultural Self Definition: Modern and Postmodern Romanian Artistic ExpressionJul 07, 2011
The scope of this analysis is to discuss the extent of change of post-communist Romania's cultural society in its self-definition, with its reclaimed national independence and its greater exposure to Western ideas, as well as the extent to which it parallels inter-war national identity developments. Some of the issues addressed include the following: How have globalization and modernization affected Romanian artistic expression in the post-1989 period? To what extent is contemporary Romanian artistic expression using the language of modernity to perpetuate old symbols of national identity? more
196. Why the Balkans?Jul 07, 2011
March 2000 - At the beginning of this new century we may ask what problems we inherited, unresolved, from the last century. One of those problems is the Balkans. more
259. Post-Communist Media Autonomy, Pluralism and DiversityJul 07, 2011
September 2002- The importance of the media is axiomatic to the new political elites in the post-Communist nations, so they seek to own, control and, at the very least, to influence the media. Consequently, Eastern Europe's media are judged to be dependent on the state - the new political forces and the newly established market - instead of being outgrowths of civil society. Furthermore, the new media systems are seen as lacking autonomy, pluralism and diversity, not contributing to the democratization process, and worse yet, being inimical to it, thwarting citizen participation. Journalism is considered unprofessional, being tendentious, opinionated, highly politicized, often inaccurate and incomplete, and pandering "to low instincts and prurient tastes."1 more
340. Acting Globally, Thinking Locally: The Side-Effects of Pursuing International Justice in the Former YugoslaviaJul 07, 2011
October 2007 - Since the end of the Cold War, there has been a marked rise in support for the international prosecution of leaders involved in some of the most heinous human rights violations. This process began with the two ad hoc international criminal tribunals for the former Yugoslavia (1993) and Rwanda (1995) and continued into the new century with the UN's Special Court for Sierra Leone (2002) and efforts to launch similar international prosecutions in other states such as Cambodia (2003) and Iraq (2005). In all but the latter case, criminal prosecutions were launched by international authorities relying on the non-coerced cooperation of leaders in sovereign states. As a result, transitional justice has become an important issue in these countries' bilateral and multilateral relations. The establishment of the International Criminal Court in 2003 suggests that the international diplomacy of transitional justice is not merely a fad but instead may become a staple. more
44. Populations and Powderkegs: The Macedonian Census of 1994 in Historical PerspectiveJul 07, 2011
The extraordinary census of the summer of 1994 provides an opportunity to view both the complexity of the Macedonian scene, of which the Albanians are a part, and the role of European mediation more broadly. The 1994 Macedonian census raises fundamental issues of which the more recent conflicts such as those over education and language use at the federal level are continuations. It is also worthy of a more detailed account as a historical moment around which national and international tensions crystallized. As this paper finds, regardless of what the future holds for Macedonia, the 1994 census is one of the key links in the chain of events leading to that future. more


text:
News online - current lead stories
us on and
Top 20 stories - for full
contents see: Statewatch News
online or What's New:
lists all items on the website.
May moves to give police powers to identify internet users: Lib
Dems welcome plan to force internet service providers to keep
details linking IP addresses to individuals (Guardian, link),
Internet data plan back on political agenda (BBC News, link) and
Security Bill will force online service providers to keep log
of users' activity (The Independent, link). The government is to
add clauses to this effect to the Anti-Terrorism and Security
Bill before parliament.
Tony Bunyan,
Statewatch Director, comments: "The government yet again,
as it did over DRIPA 2014, is openly ignoring the judgment of
the Court of the European Union (CJEU) in April that the mass
surveillance of communications, including the internet and mobile
phones, is "unlawful". See Judgment (pdf)
See for background:
Statewatch Analyses: Mass surveillance
of communications in the EU: CJEU judgment and DRIPA 2014/RIPA
2000 in the UK (pdf) and GCHQ is authorised
to spy on the world but the UK Interception of Communications
Commissioner says this is OK as it is lawful (pdf). The latter includes
a statement by GCHQ to the Intelligence and Secuirty Committee
in 2013, The reason much of GCHQs trawling is overseas
is because:
A complete
call or message between two individuals may involve a large
number of overseas CSPs and network providers
CSPs, especially those based outside the EU, may not be obliged
to retain the CD of most relevance to the authorities. Even
if they hold the relevant data, they cannot be obliged to provide
it to UK authorities, and may be unwilling to do so voluntarily. (emphasis added, p.11)
European Parliament study: The
End of the Transitional Period for Police and Criminal Justice
Measures Adopted before the Lisbon Treaty.
Who Monitors Trust in the European Justice Area? (pdf): "The
Study argues that in light of Article 82 TFEU the rights of the
defence are now inextricably linked to the coherency and effective
operation of the principle of mutual recognition of criminal
decisions, and calls the European Parliament to request the UK
to opt in EU Directives on suspects procedural rights as condition
for the UK to opt back in measures like the European
Arrest Warrant."
UK-EU JHA OPT-OUT: Final list of measures the UK will opt-out
of and the list of measures still applicable to the EU:
OPT-OUTs: List
of Union acts adopted before the entry into force of the Lisbon
Treaty in the field of police cooperation and judicial cooperation
in criminal matters which cease to apply to the United Kingdom
as from 1 December 2014 pursuant to Article 10(4), second sentence,
of Protocol (No 36) on transitional provisions (LIMITE
doc no: 15780-14, pdf)
TO UK: List
of Union acts adopted before the entry into force of the Lisbon
Treaty in the field of police cooperation and judicial cooperation
in criminal matters which have been amended by an act applicable
to the United Kingdom adopted after the entry into force of the
Lisbon Treaty and which therefore remain applicable to the United
Kingdom as amended or replaced (LIMITE doc no: 15781-14, pdf)
36 to the Lisbon Treaty - draft Schengen Decision: political
agreement and use of the written procedure for adopting it -
draft texts for publication in the OJ in C part, for information
(LIMITE doc no: 15780-14, pdf)
EP: EU-CANADA PNR AGREEMENT to be REFERRED TO COURT? Draft
Motion for a Resolution pursuant to Rule 108(6) of the Rules
of Procedure on seeking an opinion from the Court of Justice
on the compatibility with the Treaties of the Agreement between
Canada and the European Union on the transfer and processing
of Passenger Name Record data (pdf). The parliament's plenary session
will vote on this resolution on Tuesday afternoon.
SURVEILLANCE: VODAFONE & GCHQ: Spy
cable revealed: how telecoms firm worked with GCHQ (Channel 4 News, link):
of the UK's largest communications firms had a leading role in
creating the surveillance system exposed by Edward Snowden, it
can be revealed. Cable and Wireless even went as far as providing
traffic from a rival foreign communications company, handing
information sent by millions of internet users worldwide over
to spies.
The firm,
which was bought by Vodafone in July 2012, was part of a programme
called Mastering the Internet, under which British spies used
private companies to help them gather and store swathes of internet
traffic; a quarter of which passes through the UK. Top secret
documents leaked by the whistleblower Edward Snowden and seen
by Channel 4 News show that GCHQ developed what it called "partnerships"
with private companies under codenames. Cable and Wireless was
called Gerontic."
members under police surveillance mount collective legal challenge (NUJ, link): Six NUJ
members have discovered that their lawful journalistic and union
activities are being monitored and recorded by the Metropolitan
Police. They are now taking legal action against the Metropolitan
Police Commissioner and the Home Secretary to challenge this
ongoing police surveillance. The NUJ members involved in the
legal challenge include Jules Mattsson, Mark Thomas, Jason Parkinson,
Jess Hurd, David Hoffman and Adrian Arbib:
EU: Supra-national
border guard system on EU radar (euobserver, link): "The final
phase is the most far-reaching. Also known as full integration
at EU level, it entails setting up an entirely new agency
- the committee on Schengen border management (CSBM). The new
body would be composed of border guards under a EU-level command
structure. The study notes that all border guards, previously
acting under the command and control of national authorities,
would now form part of the European border guard corps."
and see European Commission: DH Home: Study: Study
on the feasibility of the creation of a European System of Border
Guards to control the external borders of the Union ESBG (pdf)
EU: Council of the European Union: Data Protection:
DP Regulation:The
one-stop-shop mechanism (LIMITE doc no: 14788-rev1-14,
68 pages, pdf)
DP Directive on LEAs exchange
of personal data between member states: DIRECTIVE
processing of personal data by competent authorities for the
purposes of prevention, investigation, detection or prosecution
of criminal offences or the execution of criminal penalties,
and the free movement of such data - Chapters I, II and V
(LIMITE doc no: 15659-14, 71 pages, pdf) Here one option for
the Council is to extend the scope from policing and law and
order to include "internal security" as well - which
could embrace internal security agencies too.
Council of the European Union: Cyber Defence Policy and Presumption
of Innocence
Cyber Defence Policy Framework (LIMITE doc no: 15193-14,
pdf): "Cyberspace is often described as the fifth domain
of military activity, equally critical to European Union (EU)
Common Security and Defence Policy (CSDP) implementation as the
domains of land, sea, air, and space. The successful implementation
of CSDP has been increasingly dependent on the availability of,
and access to, a secure cyberspace. Robust and resilient cyber
defence capabilities are now required to support CSDP structures
and CSDP missions and operations."
for a Directive of the European Parliament and of the Council
on the strengthening of certain aspects of the presumption of
innocence and of the right to be present at trial in criminal
proceedings - General approach (EU doc no: 15325-14, pdf) "This
general approach will then constitute the basis for negotiations
with the European Parliament."
USA: Senate
Blocks NSA Phone Records Measure (ABC News, link): "The Senate
on Tuesday blocked a bill to end bulk collection of American
phone records by the National Security Agency, dealing a blow
to President Barack Obama's primary proposal to rein in domestic
surveillance." And: NSA
Reform Bill Dies As Republicans Hype Threats From Islamic State (The Intercept, link)
WELCOME TO "1984": European Parliament: Automated
taxis and delivery by drone: welcome to Europe's high-tech future (Press release, pdf):
technologies such as wearable computers and face recognition
promise to change the way we work, shop and entertain. Creating
a digital single market could help boost Europes high-tech
businesses and create more jobs. At a workshop organised by the
EP's internal market committee on Thursday 13 November, MEPs
and experts looked at ways how to achieve this....
The event,
opened by R Thun, a Polish member of the EPP group, started with
a glimpse into the future. Using smart glasses, you can hail
a driverless taxi with a simple wink of the eye. Once you are
in the car, it recognises you and plays your favourite song.
It drives you to a shop where you pick what you want and walk
out. The money is deducted automatically using face recognition
technology. Or you shop online and your purchase is delivered
by a drone."
Council of the European Union developing its negotiating position
on: Anti-discrimination Directive, European Public Prosecutors
Office and Eurojust
for a Council Directive on implementing the principle of equal
treatment between persons irrespective of religion or belief,
disability, age or sexual orientation (LIMITE doc no:
14862-14, 35 pages, pdf)
for a regulation on the establishment of the European Public
Prosecutor's Office (LIMITE doc no: 14710-14, 47 pages,
pdf): "After having discussed the file during 7 working
days in COPEN/Friends of Presidency, three times in CATS, as
well as at the informal ministerial meeting in Milan in July
and in JHA Council of 10 October, the Italian Presidency now
proposes a consolidated redraft of the first 33 Articles of the
for a Regulation on the European Union Agency for Criminal Justice
Cooperation (Eurojust) - Revised text for Partial General Approach
(LIMITE doc no: 15260-14, 83 pages, pdf): "Delegates
will find attached in the Annex a further revision of the Commission's
proposal which the Presidency considers to constitute the basis
for the partial general approach at the forthcoming JHA Council."
want to see us drown" Survivors of a push back operation
in the Aegean Sea report to the Watch The Med Alarm Phone (Watch the MED, link):
"Witness Mr D. reports
of a push-back operation by the Greek coastguard when he and
32 other passengers, all of Syrian nationality and including
a pregnant woman, were leaving Cesme in Turkey to reach the Greek
island of Chios on a rubber vessel in the night of the 25th-26th
of October 2014. The Greek coastguard intercepted the vessel
and later boarded it, then took away the gas tank of the engine
and punctured the vessel. The coastguard left the vessel behind
in Turkish waters, without an engine and a hole in the vessel.
The passengers were able to call the Turkish coastguard which
rescued them and brought them back to Cesme."
See also: Safety
at sea (link) and The
sea as frontier (link)
EU: Mapping
of migrant camps
(Close the Camps, link): "A dynamic and interactive mapping
of migrant detention in Europe and beyond. This site aims to:
Record the sites, forms and conditions of migrant detention and
their serious human consequences; Enable access to information
concerning migrant detention sites and contact with detained
persons; Mobilise all those who oppose migrant detention and
removal measures to protect migrants human rights)"
INTERNATIONAL PROTECTION: Council of the European Union: Proposal
for a Regulation amending Regulation (EU) No 604/2013 as regards
determining the Member State responsible for examining the application
for international protection of unaccompanied minors with no
family member, sibling or relative legally present in a Member
(LIMITE doc no: 15120-14, , pdf)
EU: Red Cross EU: Press release: Position
paper on the Right to Access to International Protection: Recommendations
of the National Red Cross Societies of the Member States of the
European Union and the International Federation of Red Cross
and Red Crescent Societies (Press release, pdf) See also: Position
Paper: Recommendations (pdf) and Legal
avenue to protection (pdf) This position paper sets forth the following
six recommendations:
"1. Ensure
that people fleeing Syria have access to asylum procedures in
the EU
2. Consider visa applications from people fleeing Syria in a
protection sensitive way
3. Facilitate family unity in Europe for people fleeing Syria.
4. Offer emergency resettlement to the most vulnerable people
fleeing Syria.
5. Review refugee status determination procedures and reception
conditions for people fleeing Syria.
6. Do not return people to Syria and its neighbouring countries."
UK: Police
covered up links with union blacklisting - Leaked
minutes show senior officer met group targeting union activists (Guardian, link): "Scotland
Yard has been accused of seeking to cover up its involvement
in the blacklisting of more than 3,200 construction workers following
the emergence of minutes of a meeting between a senior officer
in its anti-extremism unit and the organisation running the list.
The leaked document proves that as late as 2008 a detective chief
inspector in the National Extremism Tactical Coordination Unit
(Netcu) briefed members of the Consulting Association, the secretive
organisation that ran the blacklist keeping people out of work
for decades."
SPAIN: Statewatch Viewpoint: Life imprisonment
in Spain: An inhuman and unlawful punishment (pdf) by Julián
Carlos Ríos Martín
(Professor of Penal Law at the Universidad Pontificia Comillas,
EU: Council of the European Union: DATA PROTECTION REGULATION:
Council developing its negotiating positions: Public
sector - Partial General approach (LIMITE doc no: 15389-14, 13.11.14,
pdf): "The proper functioning of the internal market
requires that the free movement of personal data within the Union
should not be restricted or prohibited for reasons connected
with the protection of individuals with regard to the processing
of personal data." [emphasis added]
IX: Provisions relating to specific data processing situations
(LIMITE doc no 15544-14, 14.11.14, pdf): With Member State positions.
delegation: To: Working Group on Information Exchange and Data
Protection (DAPIX): Subject: General Data Protection Regulation
- Processing for purposes of social protection (LIMITE
doc no: 15106-14, 5-11.14, pdf) and German
delegation: Subject: General Data Protection Regulation - Processing
in the employment context (LIMITE doc no:15108-14, 5-11-114,
EU: Council of the European Union: NIS, Migratory Pressures,
CT internal-external, ISS, Law Enforcement Working Party and
CSDP missions
NIS TRILOGUE: Proposal
for a Directive of the European Parliament and of the Council
concerning measures to ensure a high common level of network
and information security across the Union - Preparations for
the 2nd informal trilogue (LIMITE doc no: 14850-14, 4-11-14,
, pdf) Multi-column documents showing different positions of
Commission, Council and the European Parliament
Action on Migratory Pressures - A Strategic Response 5th Biannual
Update (43 pages, LIMITE doc no: 15248-14, 10-11-14,
pdf): Detailed operational plans.
of expert groups and networks related to the LEWP and the provisional
planning of their meetings (LIMITE doc no: 8709-14, 3-11-14,
pdf) Future plans:including: "2 Action Days in autumn
2014 (counter terrorism action day and metal/copper theft)"
paper on further enhancing the links between internal and external
aspects of counter-terrorism: strengthening the synergies between
TWP and COTER (LIMITE doc no: 14081-14, 13-11-14, pdf):
"The Council Secretariat, EEAS, EU CTC, CION and IntCen
should continue to highlight the relevance of specific cross-cutting
topics to groups, ensuring that coordination is enhanced and
duplication avoided where possible. This will also further encourage
a comprehensive approach to CT in the EU, ensuring that links
are made between relevant working groups, documents shared and
consultation given where relevant."
Council conclusions on the development of a renewed European
Union Internal Security Strategy (11 pages, LIMITE doc
no 14186-rev6-14, 13-11-14, pdf):
"A European PNR is one of several important tools
in this field as well as the full implementation of the Prüm
decision. Interoperability of different systems, enhancing
and simplifying existing tools should be ensured to allow a more
efficient and proportionate exchange of information and subsequently,
to enable joint operations as well as to gather evidence for
prosecution. The possible introduction of a European Police
Record Index System (EPRIS) could be explored. A framework,
respecting fundamental rights, for swift cooperation by investigating
and prosecuting authorities in access to electronic evidence
across jurisdictions held by players in industry is required,"
[emphasis added]
European External Action
Service (EEAS): CivOpsCdr
Operational Guidelines for Monitoring, Mentoring and Advising
in Civilian CSDP missions (41 pages, LIMITE doc no: 15272-14,
7-11-14, pdf)
Ministers sign agreement to share crime scene DNA (pdf): ""Australia
has joined the International DNA Search Request Network
(SRN), which makes it easier to check DNA from unsolved cases
against profiles held in another country.... Australia will join
the UK, USA and Canada in the SRN. This will give UK police access
to around 20 million profiles on three continents via a secure
Interpol network." And see: Interpol Factsheet: DNA
EU: Court of Justice of the European Union (CJEU): According
to Advocate General Sharpston, non-combat military personnel
may claim asylum if they consider themselves to be at risk of
prosecution or punishment for refusal to perform military service
where so doing might involve commission of war crimes (Press release, pdf)
and Opinion (pdf) see also: Military
deserters may claim EU asylum (euobserver, link)
See: Resources for researchers:
Statewatch Analyses: 1999-ongoing
SECILE Project:
stock of EU Counter-terrorism policy and review mechanisms: Summary
of Statewatchs findings for SECILE project (pdf)
- Catalogue
of EU Counter-Terrorism Measures Adopted since 11 September 2001 (pdf) by Ben Hayes &
Chris Jones
- Report
on how the EU assesses the impact, legitimacy and effectiveness
of its counterterrorism laws (pdf) by Ben Hayes & Chris Jones
- Data
Retention in Europe: A Case Study (pdf) by Chris Jones & Ben Hayes
EU: Borderline:
The EU's New Border Surveillance Initiatives: Assessing the Costs
and Fundamental Rights Implications of EUROSUR and the "Smart
Borders" Proposals (pdf) A study by the Heinrich Böll Foundation.
Written by Dr. Ben Hayes and Mathias Vermeulen: "Unable
to tackle the root of the problem, the member states are upgrading
the Unions external borders. Such a highly parochial approach
taken to a massive scale threatens some of the EUs fundamental
values - under the pretence that ones own interests are
at stake. Such an approach borders on the inhumane."
20th Anniversary Conference, June 2011: Statewatch
conference speeches
TNI - Statewatch:
'policy laundering' and the FATF - legalising surveillance, regulating
civil society
(pdf) by Ben Hayes
Statewatch publication:
to EU decision-making and justice and home affairs after the
Lisbon Treaty
(pdf) by Steve Peers, Professor of Law, University of Essex,
with additional material by Tony Bunyan
Neoconopticon: the EU security-industrial
(pdf) by Ben Hayes
The Shape of Things to Come (pdf) by Tony Bunyan
Since its launch this website
has had 14,511,416 user sessions and 104,056.597 "hits"
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text:
Ceremony & Vows, Etiquette
Things to Remember If Your Officiant Is Coming in from Out-of-Town
Tips for being the best host ever for your wedding officiant.
Tips for being the best host ever for your wedding officiant.
This couple met on the water, so they headed to the Rhode Island coast for their elegant early summer affair.
We all know you love him, but everyone needs a little reinforcement when it comes to the big "I do."
It's Thanksgiving — which means this might be the first time you're meeting your future husband's hometown buddies.
They have all of the best-kept secrets!
Make your reception chairs extra special by adding a sweet sign.
Plan your honeymoon before you start planning for your wedding? Yep, and here's why.
Don't wake up with regrets after the wedding about how you might have done things differently.
Find out where the couple got the name Esmeralda Amada from, and more adorable details about her life as a new mom.


text:
See our store section for gifts by different stores
See our top gift ideas section for most popular items.
Create your own personal gift registry. Register for any item, from any store, for any occasion. Find the gift registries of your family and friends.
Ever find the perfect gift idea for someone, but their birthday was months away? We make it easy to remember your gift ideas.
Provides timely email reminders for a birthday, anniversary, and other special occasions.
Get advice from friends and family on which gifts to buy.
Gift Boxes
Snacks & Munchies
Two Trays of Red Grapefruit
Fruits, Cheeses and Snacks Gift Tower
Four Caramel Pretzels and 20 Hand-Dipped Cherries
Sea Salted Caramels and Hand-Dipped Cherries
Four Caramel Pretzels and Half Dozen Champagne Strawberries
Sea Salted Caramels and Half Dozen Champagne Strawberries
Half Dozen Double Hand Dipped Chocolate Strawberries
Chocolate Dipped Cookies and a Dozen Milk and Dark Strawberries
Cheesecake Trio and Half Dozen Chocolate Strawberries
Simply Fresh Fruit Gift Box
Dried Fruit and Mixed Nuts Gift Box
Red Grapefruit Gift Box
Hand Dipped Chocolate Bananas
Organic Dried Fruit and Nut Tray with Personalized Ribbon
A Spread to Share Fruit and Snacks Gift Basket
Cheesecake Trio and Chocolate Sports Strawberries
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Sleep-In Treat Gourmet Gift of the Month Club
David's Fruit and Snack Gift Tower
Simply Fresh Fruit and Snacks Gift Basket
Fresh and Dried Tropical Fruit Gift Baskets
Deluxe Organic Fruit Gift Basket
Valencia Oranges 8 Lbs.


text:
5 Ft Dual Patch Cable Male RCA To Male 1/4"
High-quality,highly flexible cable provides L/R unbalanced audio channels,terminated with male RCA phono connectors on one end, and 1/4" at the other. 5' length.
Login Here to rate and review this product.
101-013 - 2 Mono 1/4 Inch Phone Jack
Mounts in single 3/8" hole, in panels up to 1/8" thick
Available in open body
Mfr #: 12A
27-1025 - 1/4 in F to 1/4 in F Stereo Adaptor
1/4" female to 1/4" female
Plastic case
24-13966 - 6Ft Dual RCA-M To Dual 1/4
Cable Length: 1.8mCable Length - Imperial: 5.91ftCable Length - Metric: 1.8mColour: BlackConnector Type A: 2 x Phono PlugsConnector Type B: 2x 6.35mm Mono Jack Plugs
24-12179 - 10Ft Stereo Y-Cable 3.5mm TRS To Dual 1/4" TS
Stereo Breakout, 3.5MM TRS to Dual 1/4" TS - 10'This cable is designed to connect an audio device with a mini stereo phone output to pro audio gear with unbalanced phone inputs. It is ideal for connecting an iPod®, laptop, or similar device to a mixing console.
21-1040 - Rosin Core Solder - 60/40 Tin/Lead - 1LB
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Ideal for general purpose electronic repair and assembly use. Tin/lead formulation provides the lowest possible melting point.
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text:
Welcome To GMTruckClub.com!
The #1 Chevy Truck Forum Online
Online since 2004, we're the #1 Chevy Truck & SUV user community. If you have any questions about your Chevy or GMC Truck, SUV or Crossover, or just want to connect with other GM owners and enthusiasts around the world, you've found the best place on the internet to do that.
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Registering is Free and Easy! Do it today and we'll see you on the forums soon!
Audio Amp wire harness coverter kit for stock radio
Discussion in 'Audio, Video & Gadget Tech' started by Chevy08Chase, Jul 13, 2012.


text:
Life cycle exercises for student faculty members at CU include:
- Create and approve a position
- Hire
- Change in salary
- Terminate
Employee Services will close its offices at 3 p.m. on Wednesday, Nov. 26, and will reopen at 8 a.m. on Monday, Dec. 1. Our team will respond to all inquiries upon our return.
Please note: If you're an employee who is paid monthly, your pay will arrive in your account on Friday, Nov. 28.
Have a safe and happy Thanksgiving!
Life cycle exercises for student faculty members at CU include:
1800 Grant St., Suite 400 | Denver, CO 80203 | Campus Box 400 UCA
General: 303-860-4200 | Fax: 303-860-4299 | Email: email@example.com


text:
Helpfull Things
These should help you during your game : )
Move Modyfier - This will change the moves of the pokémon who is first in your party.
Move slot one - 01xxE1DC
Move slot two - 01xxE2DC
Move slot three - 01xxD3DC
Move slot four - 01xxD4DC
Here are a few moves and there codes (put instead of the xx)
Aeroblast - B1
Hydro Pump - 38
Flamethrower - 35
Perish Song - C3
Spark - D1
Thunder - 57
Thunder Punch - 09
Items sold at Pokémarts that they don't sell, (again replace the xx with the code)
MasterBall - 01
Fire Stone - 16
Thunder Stone - 17
Water Stone - 18
Leaf Stone - 22
Rare Candy - 20
Nugget - 24
The code for these is 91xxF2D0
Masterballs in slot one of your ball pocket
Added by: Giner_1988 Jun 29th 2006, ID#4229
Who's Playing
Game Wiki
Added 30th Jun 2014, ID #409322
Added 31st May 2014, ID #391563
Added 26th May 2014, ID #389264
Added 31st Jan 2014, ID #350105
Added 9th Jan 2014, ID #341744
Added 21st May 2012, ID #144085
© Web Media Network Limited, 1999 - 2014. This site is not affiliated in any way with Microsoft, Sony, Sega, Nintendo or any video game publishers.
Privacy Policy | Terms of Service.
Super Cheats is an unofficial resource with submissions provided by members of the public. These are not usually tested by us (because there are so many), so please use them at your own risk.


text:
"I'm I the only one who just can't see the old lady.. "
I also haven't found her, but I was told there is one hiding ... let me know when you found her ;-)
I was also sad when APP was stopped, there was a lot of interesting stuff and a lot coming up, neuronal networks from a PHD student for example and last but not least your chess playing FPGA (what are your plans there). I think Big Max did an outstanding job on APP and I guess without him it wouldn't have even existed. We should be all very thankful for all the friendships he built with his work on APP. So thank you again, Max.
Cheers, Tobias
Book Review: Deadly Odds by Allen Wyler
Max Maxfield 11 comments
Generally speaking, when it comes to settling down with a good book, I tend to gravitate towards science fiction and science fantasy. Having said this, I do spend a lot of time reading ...
No 2014 Punkin Chunkin, What Will You Do?
Martin Rowe 1 Comment
American Thanksgiving is next week, and while some people watch (American) football all day, the real competition on TV has become Punkin Chunkin. But there will be no Punkin Chunkin on TV ...
Making the Grade in Industrial Design
Rich Quinnell 14 comments
As every developer knows, there are the paper specifications for a product design, and then there are the real requirements. The paper specs are dry, bland, and rigidly numeric, making ...
Book Review: Controlling Radiated Emissions by Design
Martin Rowe 1 Comment
Controlling Radiated Emissions by Design, Third Edition, by Michel Mardiguian. Contributions by Donald L. Sweeney and Roger Swanberg. List price: $89.99 (e-book), $119 (hardcover).


text:
Colchester House Rental Photos and Description
2 Bedroom Camp With Views Of Lake Champlain
Wonderful 2 bedroom camp on lake Champlain. Updates to the entire property add comfort and character to this cozy getaway. Large wrap-around sun deck complete with grill and picnic table. Two bedrooms with views of Lake Champlain open up to a second story deck and furniture. Washer/dryer in unit add another element of convenience. Nice gardens and a healthy lawn invite the outside to your doorstep.
- Suitability:
- children welcome
- non smoking only
- pets considered


text:
O.J. Simpson, Christie Prody Attacked By His Daughterby Free Britney at . Comments
In case you thought his life couldn't get any sketchier, O.J. Simpson's daughter apparently went berserk, assaulting O.J. and girlfriend Christie Prody!
Cops were called to the home of the disgraced football player and Naked Gun star after he was brutally attacked by his eldest daughter, Arnelle.
"He was cut on the back of his head, blood was coming out the side of his mouth and his lip was cut," a source told The National Enquirer.
OJ Simpson was feuding with Arnelle Simpson about her mom, Marquerite - OJ's first wife, who has been working at Walmart just to make ends meet because OJ gives her no money. OJ does support girlfriend Christie Prody, natch.
As tension mounted, Arnelle lashed out at OJ for ditching Marquerite, his high school sweetheart, to marry Nicole Brown, whom Simpson later killed.
He was acquitted of Nicole's murder in 1995, although he was found liable for her death in a civil suit a year later. Makes perfect sense, we know.
In a rage, Arnelle shoved O.J. violently into a glass cupboard. Christie Prody tried to intervene and she was subsequently attacked as well."O.J. Simpson hit the cupboard hard and the glass shattered all over him. He fell to the floor, bleeding," said the source.
Arnelle, in a moment of guilt, dialed 911 but panicked and hung up.
Despite that, police cars were dispatched to O.J.'s house in Kendall, Fla., near Miami, after the call was tracked to his address.
According to the report, O.J. Simpson - who is facing trial for robbery and kidnapping later this year - told cops he didn't want to press charges. We're surprised he didn't try to portray himself as the victim ... even if he was.
Chad, you should learn to SPELL before you criticize someone or their lifestyle!
Hes in jail now where he should been years ago.
Black people need to start acting like humans and not dogs.
I tired about reading about some black over paied stars not acting like a human. Michiel vic Jackson Oj Plexco Burrie ocho chad what ever his name is. West at the MTV awards.
Sarena willaimas, Michiel Jordon
Pack man Jones .
This just the list i have in my head i shure i can add more
Start taking these peoples jobs away see where they end up.
grow the F up tards
You people need to let that go, HE WAS ACQUITTED, so therefore, he didn't kill her!!!! We all reap what we sow, wonder what she was sowing???? hmmmm!!!!!!!
She doesn't look all that gross either!
Now, now, I'm sure she works very hard at getting it HARD!
This lady is obviously a hanger on just in this situation to see what she can get. she should move on an get a job at walmart I am sure she never worked a hard day in her life.
Ya Im Sure ^You^ Look Just As Gross Your Just Jelous OJ Aint Supporting You!!
she looks gross
This family is crazy as hell


text:
Change Your Clocks, Change Your Batteries.
Ladder 22 Crew Going to Work
On October 24, 2014 around 11:30am. West Berlin Fire-Rescue Ladder Co 22, assisted Voorhees Fire today on a 2 alarm apartment building fire. Great job by all to minimize the fire damage. Photo Credit by www.firstduenews.com
Late Night House Fire
On October 17, 2014 around 0400hrs members of the Berlin Fire Rescue and Berlin Township Fire Company responded for a reported house fire. Upon arrival the police notified all incoming fire department apparatus of the conditions ahead. The house was fully Involved in Fire, with residents being led to safety from the police. The fire department made a very quick stop on the fire and thankfully no injuries were reported to any fire personal, police officers or residents.The cause of the fire is under investigation.
On Sunday October 12,2014 Task Force 21-1 was dispatched to Haddon Ave and White Horse Pike for a report of a car fire. 21101 arrived on location to find a vehicle on fire. Engine 22 arrived shortly after and stretched a 1 1/3 hand line to extinguish the fire. Squad 211 backed up the engine and pulled a 2nd hand line. Fire was turned over to Berlin PD and the Local FM.
Fire Saftey Day!
Apartment Fire in Winslow Twp
Never Forget 9-11-01
MVA with Entrapment
On Saturday September 6,2014 Engine 22 along with Utility 22 were dispatched to the White Horse Pike in Berlin Boro for a MVA with entrapment. Crews assisted Rescue 21-1 and Rescue 232 on removing the patient from the vehicle. Accident is under investigation with Berlin Police Department. Photos credit Berlin Police Department.
Ice Bucket Challange
Another call for help and the members of West Berlin Fire Rescue answered the call. Doctor H , who is battling this horrible disease, reached out to Chief Jackson to ask for help doing his ice bucket challenge, the Chief and members stepped right up to help, as always. We're in for the fight with you Doc, you got this.
Apartment Fire in Lindenwold
On Wednesday August 6, 2014 around 7:30pm Engine 22 along with Rescue 22 were out doing weekly training on rescue operations when Engine 22 was dispatched to the 500 block of East Gibbsboro Road in Lindenwold for a report of Apartment Fire with people trapped. Engine 22 arrived first on location with smoke showing from the apartment. Crews operated with a 1 3/4 hand line extinguishing the fire quickly. Crews remained on location for about 35 minutes. Good work by all.


text:
Guides & F.A.Q's
New ThreadMercedes Threads
Just came back to maple need some help
Last: wickedtmageHey guys. Just got back into maple after quitting about 2-3 years ago. I'm level 153 and I got 10b to fund. I'm not looking to be godly. Just playing this because it's the highest level I got and i'm too lazy to restart. Below is my current damage and what items I have. Can you guys help me deci
% boss and attack
Last: drakenI have around 220% boss on my Merc from my wep, arrows, cra, and gollux, I have 30% boss 15% pdr on my unique arrows but found 85% boss arrows (25% boss neb) in the market (7 bil). Should I get them or do I have too much % boss already and should recube for % attack? I have no %attack yet on potenti
Rising Rush Problems
Last: ElufuWhenever I use rising rush, I'm usually damaged by monsters. This is because whenever I rush, I usually end up in the same exact place that the monsters are, and right before they are launched into the air, I take damage. After about 30 rushes, I have to drink a potion or I die. Is there a way to f
What to upgrade?
LockedLast: LokiBareHere are my current equips: http://i.imgur.com/dmLKz6n.jpg I don't know if I want to buy something already cubed because I'm thinking of buying cubes for black friday or cyber monday, or if there's a miracle time event. I've got about 7b right now, but might be able to raise a few more in a few
What should i improve on?
Last: RheeBreezyWeapon Pendant Ring 1,2,3,4
Help funding a mercedes
Last: yondaime555I really like mercedes play style and am going to try to fund one. I'm not trying to be the godliest mercedes around, just a decent one. I've never really funded anything other than a dual blade so I have no idea what I should be trying to get. I'm thinking about pumping 30b or so into this chara
Rising Rush change in action post Black Heaven patch in kms
LockedLast: Elufuhttps://www.youtube.com/watch?v=jamnF6IdWpw Rising Rush looks a lot like Final Toss on Aran and Upper Stab on Dual Blade. This video also shows Aerial Barrage and Rolling Moonsault being used together as well. The delay decrease on those two skills is looking good. Shame I don't play my Mercedes a
Kmst 1.2.513 - Mercedes Queen of minor patches
Last: ranixinSummary: Looks like the chaining mechanics has received a fair bit of criticism (in particular with the discomfort/clunky-ness?) and they're looking to address this with this patch. Also some mobbing damage increase here and there and .. probably the most important thing to note is.. Wrath of
Dbg nx Equip Effects
LockedLast: BlueLightI've been looking for a nice weapon cover for my Mercedes, and find it unfortunate that Ishtar's animation seems to override most nx attack effects. Are there any nx covers that retain a significant effect even during our main attacks? Or just share what you like to use for your merc.
So I kinda deleted my perfected Dbg 8 star And need a P/c
LockedLast: TheNobleRoseI usually make my own items but I don't want to relive the anger, so I'm buying one. I would like a price check on this DBG in broa please, looking to buy. Heads up everyone please lock your items :) https://imagizer.imageshack.us/v2/326x435q90/538/ASMibe.jpg https://imagizer.imageshack.us/v2
Your Thoughts on Mercedes Compared To Other Bowmen Classes
LockedLast: juubeeI want to know how think of Mercedes compared to the other bowmen classes after the latest Kms changes to her. Is she weaker or stronger than them, Or does she excel in one area or another compared to them? As your reason why. Plz and thx you.
Merc funding?
LockedLast: RavennaHello basi! I kind of need to settle down and choose a main (kind of hard for me :() and i was thinking merc couse theyre one of my favorite classes, but i have some doubts concerning its dmg and survivavilty later in the game. Can you guys help me out and explain to me how mercedes handles on those
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