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EFCC Releases Full Statement On Naira Marley's Arraignment
Azeez Fashola, aka Naira Marley, was arraigned at the Federal High Court in Ikoyi, Lagos this morning to face 11 charges bordering on fraud against him for alleged credit card fraud.
He was remanded in EFCC custody until Might 30th pending his bail software.
Learn assertion from EFCC as sighted by MandyNews.com.
"The Financial and Monetary Crimes Fee, EFCC, Lagos Zonal workplace, on Might 20, 2019, arraigned a Nigerian artiste, Azeez Adeshina Fashola, also referred to as Naira Marley, earlier than Justice Nicholas I. Oweibo of the Federal Excessive Courtroom sitting in Ikoyi, Lagos on an 11-count cost bordering on cybercrime.
The defendant was arrested on Might 10, 2019, following intelligence report obtained by the Fee about his alleged involvement in web fraud.
One of many counts reads: "That you simply, Azeez Adeshina Fashola, aka Naira Marley, and Yad Isril (nonetheless at giant), on or concerning the 26th day of November, 2018, inside the jurisdiction of this honourable courtroom, conspired amongst your self to make use of Entry card no : 5264711020433662 issued to individuals apart from you in a bid to acquire achieve and also you thereby dedicated an offence opposite to Part 27(1) (b) of the Cybercrime Prohibition, Prevention and many others Act 2015 and punishable underneath Part 33(2) of the identical Act."
The defendant pleaded not responsible to the costs most popular in opposition to him.
In view of his pleas, the prosecution counsel, Rotimi Oyedepo, requested the courtroom for a trial date.
He additionally prayed for accelerated listening to of the case and urged the courtroom to remand the defendant in jail custody.
Counsel to the defendant, Taiwo Oluwa Ore- Agba, requested the courtroom for a date to listen to the bail software on behalf of his consumer.
Consequently, Justice Oweibo adjourned the case to Might 30, 2019, and ordered the defendant to be remanded in jail custody pending the listening to of the bail software"
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text: Who do you think is having a better grip at the world right now. Illuminati, the new world order or the Rothchilds/Rockefellers? Faraz Noor khurram over 1 year ago Inhumanity does. No matter what group or religion the person belongs too. 90 0 Report postForward the question Forward the question FacebookTwitterVK

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Hypoxia is a common feature of solid tumours that promotes invasion and metastatic dissemination. Invadopodia are actin-rich membrane protrusions that direct extracellular matrix proteolysis and facilitate tumour cell invasion. Here, we show that CSRP2, an invadopodial actin bundling protein, is upregulated by hypoxia in various breast cancer cell lines, as well as in pre-clinical and clinical breast tumour specimens. We functionally characterized two hypoxia responsive elements within the proximal promoter of CSRP2 gene which are targeted by hypoxia-inducible factor-1 (HIF-1) and required for promoter transactivation in response to hypoxia. Remarkably, CSRP2 knockdown significantly inhibits hypoxia-stimulated invadopodium formation, ECM degradation and invasion in MDA-MB-231 cells, while CSRP2 forced expression was sufficient to enhance the invasive capacity of HIF-1α-depleted cells under hypoxia. In MCF-7 cells, CSRP2 upregulation was required for hypoxia-induced formation of invadopodium precursors that were unable to promote ECM degradation. Collectively, our data support that CSRP2 is a novel and direct cytoskeletal target of HIF-1 which facilitates hypoxia-induced breast cancer cell invasion by promoting invadopodia formation.
Metastasis, i.e. the spread of tumour cells from the primary tumour and subsequent colonization of distant organs, is the most life-threatening aspect of cancer1. The hypoxic tumour microenvironment is a potent driver of tumour aggressiveness and metastasis, and is highly associated with poor clinical outcomes in various cancers2,3,4. A fundamental process underlying the pro-metastatic effect of hypoxia is the stimulation of tumour cell invasive capabilities. At the subcellular level, hypoxia has recently been reported to promote the formation of actin-rich membrane protrusions, termed invadopodia5. Invadopodia facilitate tumour cell invasion through dense extracellular matrix (ECM) by recruiting transmembrane and secreted metalloproteinases (MMPs) that catalyze ECM component degradation, and creating pores through which mesenchymal tumour cells can migrate6,7. Both ex vivo and in vivo studies have provided direct evidence of the critical roles of invadopodia during key steps of the metastatic cascade, such as basement membrane breaching, intravasation and extravasation8,9,10,11,12. In addition, it has been suggested that invadopodia may contribute to other important aspects of disease progression, such as tumour growth and angiogenesis13,14, further increasing interest in their potential as therapeutic targets.
Invadopodium biogenesis largely relies on cytoskeletal rearrangements orchestrated by a combination of lamellipodial and filopodial actin machineries15,16,17,18. A critical step of invadopodium initiation is the assembly of an actin core by the ARP2/3 complex and its associated regulators, such as N-WASP and cortactin. Invadopodium elongation is promoted by the expansion of the actin core in both branched networks and unbranched bundles. At the tip of invadopodia, actin bundles presumably potentiate the protrusive force generated by actin polymerization, whereas the dendritic actin network progressively expands to fill and stabilize upstream regions16,18. The actin cytoskeleton proteins and upstream signalling pathways involved in invadopodium biogenesis have been characterized to a great extent7. However, our understanding of how important components of the tumour microenvironment, such as hypoxia, shape the invasive behavior of tumour cells remains fragmented5,7.
Cysteine-rich protein 2 (CSRP2) is a short (21 kDa) two LIM domain-containing protein, which is upregulated in invasive breast cancer cells, and localizes along the protrusive actin core of invadopodium19. Similar to its relatives CSRP1 and CSRP3/muscle LIM protein20,21, CSRP2 crosslinks actin filaments in stable bundles, suggesting that it contributes to the assembly and/or maintenance of the invadopodium actin backbone19. Accordingly, CSRP2 knockdown significantly inhibits invadopodium formation in aggressive breast cancer cells, as well as MMP secretion and 3D matrix invasion. It also strongly reduces tumour cell dissemination in two mouse models of breast cancer metastasis. The clinical relevance of these findings to human breast cancer disease is supported by microarray data identifying CSRP2 in a cluster of 14 upregulated genes characteristic of the highly aggressive basal-like breast carcinoma subtype22. In addition, among basal-like tumour patients, those with high CSRP2 expression exhibit an increased risk for developing metastasis. In the present study, we show that hypoxia upregulates CSRP2 in different breast cancer cell lines, and that such upregulation results from HIF-1-mediated transactivation of the CSRP2 promoter. We provide evidence that CSRP2 depletion strongly reduces the ability of hypoxia to enhance invadopodia formation, ECM degradation and invasion in highly invasive breast carcinoma cell lines, such as MDA-MB-231 and mouse 4T1. In weakly invasive, epithelial-like, MCF-7 cells, hypoxia-induced CSRP2 expression was required for the formation of invadopodium precursors, which were unable to promote ECM digestion due to the lack of MT1-MMP expression. Finally, we found that CSRP2 up-regulation correlates with hypoxic regions in both pre-clinical and clinical breast tumour specimens, and is associated with poor prognosis in breast cancer patients. Overall, our data point to an important role for CSRP2 in facilitating the pro-invasive and -metastatic effects of hypoxia in breast cancer.
Hypoxia promotes HIF-1 dependent CSRP2 up-regulation in breast cancer cells
The hypoxic tumour microenvironment is a critical promoter of breast cancer progression and metastasis3,23. We assessed the effects of hypoxia on the expression of the pro-invasive and -metastatic invadopodial protein CSRP2 in four breast cancer cell lines, including luminal/epithelial-like MCF-7 and T47D (ER+, PR+), and mesenchymal-like MDA-MB-231 and Hs578T (ER−, PR−, HER2−, claudin-low). In agreement with our previous report19, CSRP2 was absent or only weakly expressed in epithelial-like cells under normoxia, whereas it was expressed at significant levels in mesenchymal-like cells (Fig. 1A and B). Exposing cells to hypoxia (0.1% p02) for 24 hours induced a significant up-regulation of CSRP2 in all four cell lines (Fig. 1A). Indeed, CSRP2 protein levels increased by about ten times in epithelial-like cells and by about five times in mesenchymal-like cells, as compared to the respective normoxic controls. At 48 hours of hypoxia, high CSRP2 protein expression was maintained in all cell lines (Fig. 1B). To extend these data, CSRP2 transcript levels were analysed in normoxic and hypoxic conditions using real-time qRT-PCR. In the four breast cancer cell lines, hypoxia induced a significant and sustained elevation of CSRP2 transcripts (Fig. 1C and D), suggesting that CSRP2 levels were regulated at the transcriptional level.
Tumour cells respond to hypoxic stress by an activation of HIFs, which drive the transcription of target genes through binding to cis-regulatory elements, termed hypoxia responsive elements (HREs)24. Accumulating evidence indicates that HIF-1 functions as a master regulator of the hypoxic response in breast cancer2,23. To evaluate the role of HIF-1 in hypoxia-induced CSRP2 expression, its oxygen-regulated subunit (HIF-1α) was depleted in MCF-7 cells prior exposure to hypoxia. HIF-1α knockdown significantly inhibited hypoxia-induced CSRP2 up-regulation at both protein and mRNA levels (Fig. 2A,C and D). Similar results were obtained by knocking down HIF-1α in MDA-MB-231 cells (Fig. 2B,E and F), supporting that the gene encoding CSRP2 is a novel, and direct downstream target of HIF-1.
HIF-1α drives CSRP2 transcription by direct binding to HRE motifs
The consensus core HRE sequence 5′-[A/G]CGT-3′ was initially characterized based on the comparison of various known HIF target genes25. Later on, flanking nucleotides were demonstrated to contribute additional information to the likelihood of HIF-1 transactivation26. In silico analysis using the weight matrix-based program Alggen PROMO27, identified two high-confidence HREs (HRE1 and HRE2) in the proximal promoter region of CSRP2, while manual scanning found two additional lower confidence core HRE sequences further upstream (Fig. 2G).
To validate HIF-1α binding to these two putative HREs, we conducted ChIP analyses, using HIF-1α specific antibody (Fig. 2H). Results revealed that HIF-1α significantly binds to both HREs, even under normoxic conditions. Additionally, there was a dramatic and significant increase (>12 fold) in HIF-1α occupancy at these sites under hypoxia (Fig. 2H–J).
To study the transactivation of CSRP2 by HIF-1α, the nucleotides −1,920 to +103 relative to the transcription start site were cloned into a luciferase reporter vector. MCF-7 cells were chosen to conduct the reporter assays as they express low levels of CSRP2 and HIF-1α under normoxic conditions (Fig. 2A). As shown in Fig. 2K, hypoxia induced a significant increase in luciferase activity as compared to normoxia. A VEGFA promoter luciferase reporter was used as a positive control of hypoxia-induced gene transcription. Confirming the ChIP results, mutation of HRE1 decreased the hypoxic induction of transcriptional activity at the CSRP2 promoter, while mutation of HRE2 reduced this induction to an insignificant amount (p = 0.1237). These results suggest that the HRE2 sequence is the major motif responsible for hypoxia-dependent induction of CSRP2 expression, while HRE1 may also contribute to a lesser extent.
To further confirm that the CSRP2 promoter is transactivated by HIF-1α, constitutively stable HIF-1α was co-transfected into MCF-7 cells along with the luciferase reporter plasmids. Again, a VEGFA promoter luciferase reporter construct was used as a positive control of HIF-1α transcriptional activity. As shown in Fig. 2L, HIF-1α induced a significant transactivation of the CSRP2 promoter, supporting its role in hypoxia-promoted CSRP2 expression.
Taken together, ChIP and luciferase reporter experiments show that hypoxia induces binding of the HIF-1α transcription factor to the CSRP2 promoter, primarily at the HRE2 site, and subsequent induction of CSRP2 expression.
CSRP2 mediates hypoxia-stimulated invadopodium formation
The role of CSRP2 in hypoxia-stimulated invadopodium formation and activity was investigated in both a highly invasive cell line with significant basal levels of CSRP2 (MDA-MB-231), and a weakly invasive cell line with low basal expression of CSRP2 (MCF-7). First, fluorescent gelatin degradation assays were conducted with MDA-MB-231 cells following transfection with control scrambled siRNAs (siCtr) or CSRP2 targeting siRNAs (siCSRP2 #1 and #2; Fig. 3A). As illustrated in Figs 3B and S1, hypoxia promoted invadopodia-mediated ECM degradation in siCtr-transfected cells, as shown by a significant overall increase in the density of dark punctate in the fluorescent matrix background. Quantitative analyses revealed that the percentage cells associated with local ECM degradation (% of active cells) increased from about 50% in normoxia to about 70% in hypoxia (Fig. 3C, left panel). Even more remarkably, hypoxia induced a 5-fold increase in the average surface of matrix degradation per cell (degradation index; Fig. 3C, right panel).
Consistent with these data, siCtr-transfected MDA-MB-231 cells exhibited a higher number of mature invadopodia (as defined by F-actin and cortactin co-labelled dots overlapping with areas of gelatin clearing) and were significantly more invasive in hypoxia as compared to normoxia (Fig. 3D and E, respectively). CSRP2 knockdown inhibited the stimulatory effects of hypoxia and decreased the percentage of active cells, the degradation index and the number of invadopodia per cell to values similar to those obtained for control cells in normoxia (Figs 3B–D and S1). In addition, CSRP2 knockdown inhibited tumour cell invasion under hypoxia with an almost similar magnitude as HIF-1α knockdown (Fig. 3E and F). In contrast, CSRP2 depletion did not significantly modify the MDA-MB-231 cell migration under normoxia or hypoxia, supporting that CSRP2 has a specific function in invasion (Fig. S2). Remarkably, CSRP2 forced expression was sufficient to increases the invasiveness of HIF-1α depleted MDA-MB-231 cells under hypoxia (Fig. 3G). In agreement with our previous report19, CSRP2 knockdown also repressed invadopodia-mediated matrix degradation and invasion under normoxia (Fig. 3B–E). Collectively, these data suggest that hypoxia promotes invadopodia-mediated breast cancer cell invasion by upregulating CSRP2, a basic component of the invadopodial actin cytoskeleton machinery. To strengthen this conclusion, we validated the role of CSRP2 in facilitating hypoxia-stimulated invadopodia formation, ECM degradation and cell invasion in another invasive breast cancer cell line, namely the mouse 4T1 cell line (Fig. S3).
Contrary to MDA-MB-231 and 4T1 cells, MCF-7 cells failed to promote localized gelatin degradation under both normoxia and hypoxia (Fig. 4A). This lack of activity was consistent with the fact that MCF-7 cells did not express membrane type-1 MMP (MT1-MMP; Fig. 4B), a membrane-tethered MMP required for proteolytic activity28,29. However, hypoxia promoted cortactin relocalization to actin puncta, whose appearance and localization at the ventral side of tumour cells were reminiscent of invadopodia (Fig. 4A). Similar ventral actin/cortactin puncta were induced by hypoxia when MCF-7 cells were plated on non-denatured collagen (Fig. S4). To further examined the nature of these structures, we used two additional invadopodium markers, namely N-WASP and Tks530. Both effectively labelled the actin/cortactin puncta that formed under hypoxia (Fig. 4F and G), which were accordingly termed invadopodium precursors. As shown in Fig. 4H, CSRP2 was also recruited to hypoxia-induced invadopodium precursors, suggesting it is required for, or possibly mediates, their formation. To assess this possibility, the number of invadopodium precursors was determined in MCF-7 cells that were transfected with control or CSRP2-targeting siRNAs prior to incubation in normoxic or hypoxic conditions. Under normoxia, both siCtr- and siCSRP2-transfected cells exhibited about two invadopodium precursors on average (Fig. 4C and D). Hypoxia increased invadopodium precursors density in control cells by almost 9-fold, with an average of 17 invadopodium precursors per cell. Interestingly, this effect was associated with a significant increase in the secretion of pro-MMP-2 and pro-MMP-9, suggesting that, although proteolytically inactive, these invadopodium precursors are mature enough for MMP secretion (Fig. 4E). CSRP2 knockdown significantly lowered the number of hypoxia-induced invadopodium precursors, with about eight precursors per cell on average (Fig. 4C and D). Accordingly, it also inhibited hypoxia-promoted secretion of proMMP-2 and proMMP-9 (Fig. 4E). Thus, although MCF-7 cells lack MT1-MMP expression and matrix degrading activity, they assemble invadopodium precursors in response to hypoxia in a CSRP2 dependent manner.
Collectively our data indicate that CSRP2 an important mediator of hypoxia-stimulated invadopodium formation in breast cancer cells.
CSRP2 is upregulated in hypoxic, HIF-1α-positive regions of human breast cancer cell line xenografts
To validate that CSRP2 is up-regulated by intratumoral hypoxia, MCF-7 and MDA-MB-231 cells were orthotopically injected in the mammary fat pad of immunodeficient mice, and the resulting primary tumours were collected for immunofluorescence analyses. As shown in Fig. 5A, CSRP2 was strongly upregulated in hypoxic, pimonidazole-stained, regions of both MCF-7- and MDA-MB-231-cell derived tumours. In addition, tumour sections were co-labelled for CSRP2 and HIF-1α (Fig. 5B). Strong CSRP2 signals were very frequently associated with HIF-1α positive regions in both MCF-7 and MDA-MB-231 tumour xenografts. Pixel intensity correlation analyses confirmed a strong correlation between CSRP2 and HIF-1α expression in all tumour sections analysed, with Pearson coefficient values ranging from 0.50 to 0.85 for MCF-7 cell-derived tumours, and from 0.63 to 0.86 for MDA-MB-231 cell-derived tumours (n = 22 and 21, respectively; Fig. S5). These data provide compelling evidence that CSRP2 expression is not only stimulated by experimental hypoxia produced in low-oxygen incubators, but is also induced by in vivo tumour hypoxia.
Clinical significance of hypoxia-induced CSRP2 expression in breast cancer
We next assessed the co-expression of CSRP2 and metastasis-effector genes reported to be regulated by HIF in patient tumours31. This analysis was conducted using a publicly available gene expression database of a large cohort of breast cancer patients (n = 1,215). We found that CSRP2 expression positively correlates with the expression of 21 out of 25 genes for which the expression data is available (p < 0.01; Table 1). Correlation coefficient values >0.3 were obtained for 9 of these 21 genes, and correlation coefficient values >0.5 were obtained for 2 genes, LOXL4 (r = 0.54; p = 1.5e-92) and MET (r = 0.50; p = 1.5e-79).
To more directly evaluate CSRP2 up-regulation in hypoxic areas of human breast tumours, we conducted immunohistochemical analyses of clinical breast cancer specimens. Looking for hints of a hypoxic expression profile, we found that CSRP2 was frequently upregulated in or near the centre of tumour nests, a presumably highly hypoxic region (Fig. 5C). Next, we analysed and compared CSRP2 and HIF-1α protein expression in 48 triple negative breast cancer tissue array samples, and the results were scored by a pathologist (Table S1). 14/48 (>30%) cases showed weak to obvious HIF-1α expression, of which 11 (>78%) were also positive for CSRP2. Rank analysis confirmed a statistically significant correlation between CSRP2 and HIF-1α protein levels in clinical samples with a correlation coefficient value of 0.42 (p = 0.0027). In addition, CSRP2 and HIF-1α exhibited partially overlapping distribution in successive tumour sections (Fig. 5D). As previously reported19, CSRP2 staining was also frequently observed in inflammatory/immune cells. Such staining likely account for the lower, yet significant, correlation obtained for clinical samples as compared to mouse xenografts.
To evaluate the clinical relevance of our findings, the prognostic value of CSRP2 in breast cancer was evaluated by Kaplan Meier analysis using transcriptomics data sets including 1402 breast cancer patients32. High CSRP2 mRNA expression was significantly associated with reduced overall survival for breast cancer patients. The best cut off between low and high CSRP2 expression to discriminate the subgroups with different outcomes was determined to be the upper quartile (HR = 1.6, logrank P = 0.00011; Fig. 5E). Upper quartile survival, i.e. time beyond which 75% of the patients are expected to survive, for patients with low and high CSRP2 expression was 118.8 and 64.8 months, respectively. Stratifying patients according to upper tertile or median CSRP2 expression values established weaker, yet significant, association between CSRP2 expression and overall survival (HR = 1.49, logrank P = 0.00056, and HR = 1.28, log-rank p = 0.025, respectively; Fig. S6). In addition to overall survival, CSRP2 overexpression significantly correlated with shorter distant metastasis- and relapse- free survival (p = 0.015 and 1.9e-07, respectively; Fig. 5F and G). Comparison of CSRP2 expression in different breast cancer subtypes revealed that CSRP2 expression is an order of magnitude higher in the basal subtype, compared to the other subtypes and, accordingly, is a stronger predictor of distant metastasis-free survival (Fig. S7).
Taken all together, our data suggest that tumour hypoxia promotes CSRP2 overexpression, exacerbating the clinical outcome of breast cancer patients by enhancing tumour cell invasion and subsequent spread to distant tissues.
Both experimental and clinical studies have provided evidence that the hypoxic tumour microenvironment is an important risk factor for distant metastasis in breast cancer3. Here, we identified a novel mechanism by which hypoxia promotes mesenchymal invasion in breast cancer cells through the upregulation of CSRP2, a structural component of the actin cytoskeleton machinery involved in invadopodium formation19. Our functional investigations revealed that CSRP2 knockdown in MDA-MB-231 cells inhibits hypoxia-stimulated invadopodia formation and ECM degradation. Accordingly, it also significantly suppressed the stimulatory effect of hypoxia on MDA-MB-231 cell invasiveness. Conversely, forced CSRP2 expression in HIF-1α-depleted hypoxic MDA-MB-231 cells was sufficient to increase cell invasion to an extent similar as in control hypoxic cells. Together these data support that CSRP2 is not only required for hypoxia-stimulated invasion, but contributes to drive this process.
Contrary to MDA-MB-231 cells, MCF-7 cells were unable to carry out localized gelatin matrix degradation under normoxia or hypoxia. In keeping with this, our western blot analyses revealed that both normoxic and hypoxic MCF-7 cells do not express MT1-MMP, a membrane anchored MMP which is critically required for tumour cell proteolytic activity28,29. Although proteolytically inactive, MCF-7 cells responded to hypoxia by assembling invadopodium precursors characterized by the re localization of cortactin, N-WASP and Tks5 to actin accumulations at the ventral cell membrane. This finding is highly consistent with a previous report showing that MT1-MMP depletion in MDA-MB-231 cells strongly inhibits gelatin matrix degradation but only modestly alters the onset of invadopodium formation29. In tumour cells, soluble MMP secretion is facilitated at invadopodia33. Thus, the increased secretion of MMP-2 and MMP-9 that is associated with the formation of invadopodium precursors in hypoxic MCF-7 cells (Fig. 4E) represents another piece of evidence of the nature of these structures. Consistent with the fact that MT1-MMP catalyses MMP-2 activation by cleavage of its pro-domain34, and that MMP-2 contributes to MMP-9 activation35,36, mostly inactive, high-molecular weight, forms of MMP-2 and MMP-9 were detected in our serum-free gelatin zymography assays.
Since invadopodium assembly and activity seems to be decoupled in hypoxic MCF-7 cells, this cell line turned out to be an appropriate experimental model to further characterize the mechanism by which CSRP2 mediates hypoxia-stimulated invasion. Indeed, our quantitative data show that CSRP2 knockdown significantly reduced the number of invadopodium precursors induced by hypoxia in MCF-7 cells, validating the structural role of CSPR2 in invadopodium assembly we previously suggested19.
Our mechanistic investigations revealed that the proximal promoter of CSRP2 can be activated by either hypoxic culture conditions or HIF-1α overexpression, and that it contains two HREs (HRE1 and 2) able to promote HIF-1 recruitment. Although both HREs were found to contribute to hypoxia-induced transactivation of CSRP2 promoter, our data luciferase reporter assays suggest that HRE2 has a predominant role over HRE1. Consistent with ChIP and luciferase reporter data, CSRP2 and HIF-1α protein levels were significantly correlated in both pre-clinical and patient tumour specimens, supporting that tumour hypoxia is an important determinant of CSRP2 up-regulation in breast cancer. The four breast cancer cell lines we analyzed responded to experimental hypoxia by increasing CSRP2 mRNA and protein expression. As previously reported19, invasive cells (MDA-MB-231 and Hs578T) exhibited significant basal CSRP2 levels under normoxia while weakly invasive cells (T47D and MCF7) did not. We noticed that invasive cells frequently exhibited detectable amounts of HIF-1α under normoxia (as evaluated by western blot, Fig. 1A and B). Normoxic stabilization of HIF-1α and activation of HIF-1 signalling in triple negative breast cancer cells, such as MDA-MB-231 cells, was recently shown to be regulated by a long noncoding RNA, namely LINK-A37. This pathway may account, at least to some extent, for normoxic expression of CSRP2 in triple negative breast cancer cells, which in turn contributes to maintain a constitutive invasive phenotype19. The significant basal expression level of CSRP2 in invasive normoxic cells (Fig. 1) is not contradictory with the role of CSRP2 in mediating the pro-invasive effects of hypoxia, and shows that hypoxia operates in breast cancer cells by up-regulating an actin regulatory protein that is also required for normoxic invasion. Other hypoxia-invadopodia axes were identified in cancer of different origins, such as melanoma, fibrosarcoma, and head and neck cells38,39,40,41. To our best knowledge, our report is the first example where hypoxia promotes cancer cell invasion by direct, HIF1-mediated, targeting of a basic structural cytoskeletal component of invadopodia.
A recent study has provided evidence that pancreatic ductal adenocarcinoma (PDAC) invasion and metastasis are promoted by HIF-1 dependent upregulation of another actin bundling protein, namely fascin42. Although the effects of fascin overexpression on PDAC cell invadopodium formation were not specifically evaluated, fascin has been shown to be a critical invadopodial component in prostate cancer and breast cancer cells16,43,44. Noticeably, fascin and CSRP2 knockdown achieves comparable reduction in ECM degradation and 3-D invasion in invasive breast cancer cells16,19,43, suggesting a functional interaction/redundancy between these two actin bundling proteins. Remarkably, fascin ranks first in the most highly correlated genes to CSRP2 in invasive breast cancer tumours (publicly available TCGA data at the cBioPortal for Cancer Genomics, http://www.cbioportal.org), raising the possibility that hypoxia and HIF-1 coordinate CSRP2 and fascin upregulation to promote invadopodium assembly/stabilization and invasion.
The clinical significance of our findings is further supported by transcritomic-associated survival analysis indicating that CSRP2 overexpression is associated with significantly shorter overall, metastasis- and relapse-free survival. Besides its role in breast cancer, CSRP2 was recently identified as a downstream target of H19, the long non-coding RNA with the strongest association with colorectal cancer patient survival45. Patients with high CSRP2 expression in colorectal tumours, display significantly shorter overall survival, and combined analysis of H19 with CSRP2 appears to be a powerful prognostic factor for overall survival. Another recent clinical study established a significant association between CSRP2 expression and B-cell acute lymphoblastic leukemia (ALL) relapse, and proposed CSRP2 as a prognostic marker for B-cell ALL patients with normal cytogenetics46. Functional analyses suggest that CSRP2 promotes B-cell ALL cell proliferation, migration, and drug resistance. Because hypoxia is a common feature of the bone marrow microenvironment that promotes HIF-dependent blood cancer progression and resistance to therapy47, our data call for evaluation of hypoxia-regulated CSRP2 expression in blood cancers. Although the specific functions of CSRP2 in breast cancer, colorectal cancer and B-cell ALL may differ, these studies all point to the deleterious clinical consequence of CSRP2 overexpression.
Collectively, our data indicate that hypoxia exerts a direct control on invadopodium-mediated tumour cell invasion through HIF-1-mediated upregulation of the actin-bundling protein CSRP2 and provide a new mechanistic basis for the pro-metastatic effects of tumour hypoxia in breast cancer.
Cell lines
MCF-7 cells were purchased from ATCC. MDA-MB-231, Hs578T, T47D and 4T1 cells were available at the Luxembourg Institute of Health. These cell lines were regularly tested for mycoplasma contamination and cultured in complete growth medium following ATCC recommendations. A standard tissue culture incubator was used for a normoxic culture conditions (21% O2; 5% CO2). For hypoxic treatment, cells were placed in a hypoxia work station (Invivo2 400, Ruskinn) for 24 hours or 48 hours, calibrated to maintain a hypoxic atmosphere of 0.1% O2 and 5% CO2 by continuous flow of nitrogen. The CSRP2-depleted 4T1 and corresponding control cell lines by lentiviral transduction. CRP2 knockdown was achieved by pGIPZ lentiviral shRNAs (clone ID: V3LMM_417823 from GE Dharmacon, gene set RMM4532). A non-silencing shRNAs (RHS4346, sh-; GE Dharmacon) was used as a control. Lentivirus production was achieved by co-transfecting lentiviral pGIPZ shRNAs with packaging and envelope plasmids in HEK293T cells using Xtreme transfection reagent (Roche). 4T1 cells were infected with virus, and transduced cells were selected with 0.5 μg/ml puromycin (Sigma-Aldrich).
Western blot analysis
Total protein extracts from cells was prepared in RIPA lysis buffer (Millipore) supplemented with protease and phosphatase inhibitor mixture (Roche). The extracts were subjected to Western blot using antibodies directed against CSRP2 (HPA045617, Sigma), Actin (A2066, Sigma), HIF-1α (610959, BD biosciences), and MT1-MMP (ab51074, Abcam. Protein bands were detected using Western Lightning Ultra (Perkin Elmer) and visualized with CL-Xposure film (Thermo Scientific). Protein levels were quantified using ImageJ (NIH, Bethesda, USA).
siRNA transfection
CSRP2 knockdown was achieved by transfection of 10 nM of 2 different predesigned siRNAs directed against human CSRP2 (siCSRP2#1, SI04283727, target sequence 5′-ACAGTGGCAATTCACGATGAA-3′ and siCSRP2#2, SI04251863, target sequence 5′-ACAGGCCTACAACAAATCCAA-3′; QIAGEN) using DharmaFECTTM transfection reagent (GE Dharmacon) and following manufacturer's instructions. HIF-1α knockdown was achieved using 2 different predesigned, functionally verified, siRNAs directed against human HIF-1α (siHIF1#1, SI02664053, targeted sequence 5′-AGGAAGAACTATGAACATAAA-3′, Qiagen and siHIF1#2, targeted sequence 5′-TACGTTGTGAGTGGTATTATT; Eurogentec), while non-targeting siRNAs were used as a control (Eurogentec). Freshly transfected cells were incubated 24 h under normoxia prior subsequent treatment or analysis.
Quantitative RT-PCR
Total RNA was isolated using the miRCURY RNA Isolation Kit (Exiqon) following the manufacturer's instructions. Purified RNA was reverse transcribed to cDNA using the Reverse Transcriptase Core kit (Eurogentec). Quantitative real-time PCR reactions were performed using SYBR Green I (Qiagen) on an Applied Biosystems ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). The primers used were 5′-GATCTCGGACTCCCTGGAC-3′ (forward) and 5′-TCCCCAGACAGGCATTTT-3′ (reverse) for CSRP2 and 5′-TGTCCTGAATGTGGTCACCTGA-3′ (forward) and 5′-CTGCAGTCTCCTTGCACACCT-3′ (reverse) for mitochondrial ribosomal protein L32 (MRPL32). To calculate the relative abundance of the mRNA transcripts, the ΔΔCt method was used, with MRPL32 used as a reference mRNA. The Y-axis reflects fold change in gene expression.
Chromatin immunoprecipitation assay
MCF-7 cells were grown in 15 cm plates. Once the cells had reached 70% confluence, they were subjected to 24 hours of normoxia or hypoxia, as indicated. Chromatin was isolated and sheared using the Bioruptor Pico sonicator (Diagenode), with 10 cycles of 30 seconds on at high power, followed by 30 seconds off. Sheared chromatin was used for ChIP, with anti-HIF-1Αα (ChIP grade, Active Motif, 39665) or negative control antibodies, with the ChIP-IT Express kit (Active Motif), according to the manufacturer's protocol. Pulled-down chromatin was then purified using the Chromatin IP DNA Purification Kit (Active Motif) and RT-PCR was performed using the following primer pairs: 5′-AAGTCCCTCTCCAAGTCC-3′ (forward) and 5′-CCACCAGGAGACAAAAGG-3′ (reverse) for HRE1, and 5′-TGCCTTTTGTCTCCTGGTG-3′ and 5′-ATGGGGATGTCGGAGGAGA-3′ (forward) for HRE2.
Luciferase reporter assay
Genomic DNA was purified from MCF-7 cells, using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen). A region corresponding to −1,920 to +103 bp relative to the transcription start site of the human CSRP2 gene was PCR amplified using the primers 5′-GATCACGCGTCAGGGACAGACAGCAGAAACA-3′ (forward) and 5′-GATCAGATCTTTGAGTCGGAGGCGGGAGCACGTAC-3′ (reverse), and inserted into theMluI-BglII multiple cloning site of the pGL3-Promoter vector (Promega). The HRE1 and HRE2 sites were mutated using overlapping PCR strategy and the following primer pairs: 5′-GAGGGAGCGGTCCCGGGAGCTGGGAAG-3′ (forward) and 5′-CGCCCGGCATCTTCCCAGCTCCCGG-3′ (reverse) for HRE1*, and 5′-CTAGCTCCGCCTGCGGTAGCTGCTCC-3′ (forward) and 5′-CTCACTTGAGTCGGAGGCGGGAGCAGCTACCGC-3′ (reverse) for HRE2*. The VEGF promoter luciferase reporter plasmid was provided by Dr. Amato J. Giaccia (Standford University school of Medicine, CA, USA). Briefly, 7 × 104 cells were seeded in each well of a 24-well plate. Six hours after plating, cells were transfected with the promoter luciferase reporter constructs, pGL4.73 [hRluc/SV40] vector (which contains the Renilla luciferase sequence downstream of the SV40 promotor), and, where indicated, a constitutively stable mutant HIF-1α expression vector (Addgene ref #52636), using Turbofect (Thermo Fisher Scientific), according to the manufacturer's protocol. Immediately after transfection, the plates were placed in either a normoxic or hypoxic incubator, as indicated. Twenty four hours after transfection, firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter assay kit (Promega) and the ratio of firefly/Renilla luciferase was determined.
Cell invasion and migration assays
CSRP2- or HIF-1-depleted cells and control cells were incubated for 24 hours in hypoxia or normoxia. Fifty thousand cells were subsequently transferred onto 8.0 µm-pore transwell inserts (Greiner) in serum free DMEM, as previously described19. For invasion assays, transwell inserts were pre-coated with 100 µl of EHS matrix (330 µg/ml in serum-free DMEM) and incubated for 1 hour at 37 °C to allow polymerization. For migration assays, transwell inserts were pre-coated with collagen (100 µg/ml, collagen I, Millipore). The wells were filled with DMEM supplemented to 10% FBS as a chemoattractant. After 18 hours (MDA-MB-231 cell migration), 24 hours (MDA-MB-231 cell invasion) or 48 hours (4T1 cell invasion) incubation in hypoxia or normoxia, the total number of cells and the number of invasive cells were evaluated by MTT, and the percentage of invasion was calculated and normalized to siCtr-transfected normoxic cells (set to 1). For some specific assays, cells were co- transfected with HIF-1A targeting siRNAs and a plasmid allowing CSRP2 overexpression. The latter plasmid was generated by inserting CSRP2 coding sequence into the XhoI-BamHI cloning site of modified pCDNA 3.1- (Invitrogen) including a terminal HA tag. The primers used to PCR amplify CSRP2 coding sequence were 5′-GATCCTCGAGATGCCTGTCTGGGGAGGTGG-3′ (forward) and 5′-GATCAGGATCCCGCTGGGCATGAACAAGAGCCC-3′ (reverse). An empty pCDNA 3.1- was used as a control.
Gelatinase zymography assays
Four hundred thousand MCF-7 cells were plated in each well of a six-well plate. Six hours later, they were transfected with either control or CSRP2-targeting siRNAs. Twenty-four hours later, cells were washed with PBS, incubated with serum-free DMEM, and the plates were incubated in normoxic or hypoxic conditions for 32 hours. The conditioned media was collected and concentrated using Amicon Ultra Centrifugal Filters, 30 kDa cut-off (Merck Millipore, 803024), from an initial volume of two ml to a final volume of 22 µl. The conditioned media was then subjected to gelatinase zymography analysis. Briefly, the conditioned media was mixed with 4x sample buffer (0.25 Tris/HCl pH 6.8, 40% glycerol, 8% SDS and 0.1% bromophenol blue) and resolved on a 10% SDS-polyacrylamide gel containing 0.01% porcine gelatin (Sigma, G-8150). The MMPs were then renatured by soaking the gel in renaturing buffer (2.5% Triton in dH2O) for 30 minutes. The gels were then washed several times with dH2O and incubated for 30 minutes with developing buffer (0.05 M Tris-HCl pH 7.8, 0.2 M NaCl, 0.005 CaCl2, and 0.02% Brij-35). The buffer was then poured out, fresh buffer added and the gel was incubated overnight at 37 C. The following day, the gels were visualized with Coomassie blue staining.
Fluorescent gelatin degradation assays
Fluorescent gelatin slides were prepared similarly as described in Artym et al.29. In brief, 16-mm coverslips were coated with poly-L-Lysine (25 µg/ml, 20 minutes at room temperature) and crosslinked with glutaraldehyde (0.5%, 15 minutes at room temperature). After extensive PBS washing, coverslips were inverted on a 60 µl drop of 0.2% gelatin solution containing a mix of 1:20 Oregon green 488-labelled gelatin (Molecular Probes) and unlabeled porcine gelatin (Sigma). After a 15-minute incubation, and 3 washes, coverslips were sterilized with 70% ethanol for 30 minutes and subsequently incubated with complete medium for 30 minutes. MDA-MB-231 cells were pre-incubated for 32 hours in hypoxia or normoxia before they were transferred onto gelatin-coated coverslips for a 16-hour incubation period in the same culture conditions. A low-density seeding was used to avoid cell clustering and facilitate the analysis. MCF-7 and 4T1 cells were directly loaded onto gelatin-coated coverslips and incubated for 48 hours in hypoxia or normoxia. Then, tumour cells were fixed and stained for actin and cortactin (see "Confocal microscopy and cell imaging" subsection). Active cells were defined as cells with dark dots (corresponding to fluorescent gelatin-cleared areas) underneath actin-cortactin co-labelled invadopodia. Digested gelatin areas were quantified with the threshold tool in ImageJ software, and an average degradation index (degraded matrix area per cell) was calculated.
Confocal microscopy and cell imaging
Imaging was performed on a laser scanning confocal microscope (LSM880 FastAiry, Carl Zeiss) equipped with a x63/1.4 numerical aperture (NA) oil immersion Plan-Apochromat objective for cell imaging or a x40/1.3 oil immersion Plan-Apochromat for tumour slice imaging. All pictures were acquired with multitrack configuration with a confocal optical slice set at 1 µm thickness. MDA-MB-231, 4T1 and MCF-7 cells were plated on gelatin-coated coverslips (see "Fluorescent gelatin degradation assays" subsection) for 16 hours or 48 hours. MCF-7 cells were also plated on collagen-coated coverslips (100 µg/ml, collagen I, Millipore) for 48 hours. Pre-fixation was performed in 3.5% PFA and 0.3% Triton X-100 in PEM (100 mM PiPES, pH6.9, 1 mM EGTA and 1 mM MgCl2) for 3 minutes whereas fixation was performed in PFA 3.5% in PEM for 20 minutes. Cortactin and N-WASP were immunodetected using mouse monoclonal antibodies (clone 4F11, Millipore) and rabbit monoclonal antibodies (clone 30D10, Cell Signalling), respectively. The actin cytoskeleton was labelled using Acti-stain 488 or 555 or 670 (Cytoskeleton) phalloidin depending on the experimental conditions. To facilitate evaluation, actin cytoskeleton was always depicted in green regardless of the type of staining used. Before confocal microscopy imaging, coverslips were mounted in Fluoromount medium (Sigma). In some experiments, MCF-7 cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) with a construct coding for GFP-fused Tks5 (Tks5-GFP; kind gift from Dr. Sara A. Courtneidge, Portland, USA) or a construct coding for GFP-fused CSRP2 (CSRP2-GFP). The latter was obtained by inserting the CSRP2 coding sequence into the pEGFP-N1 plasmid vector via XhoI and BamHI restriction sites. After 48 hours in hypoxic conditions, MCF7 cells expressing CSRP2-GFP were incubated 4 hours with live cell imaging actin probe (siR-Actin 1 µM, Cytoskeleton) before observation under the microscope in hypoxic conditions (5% CO2 and 0.1% O2).
In vivo xenograft assays and immunofluorescent staining
Animal work was conducted in accordance with the national and international regulations. The protocols were reviewed and approved by the animal welfare body of LIH (protocol LECR-2016-07), and received an authorization from the Ministry of Agriculture and Ministry of Higher Education and Research. Five million of MCF-7 or MDA-MB-231 tumour cells in 50 µl matrigel diluted 1:1 in PBS were subcutaneously implanted into the mammary fat pad of NOD scid gamma (NSG; NOD.Cg-Prkdcscid Il2rgtm1WjI/SzJ), 8-week-old female mice (Charles River). Primary tumour dimensions (length, width and height) were periodically measured using calipers and the tumour volume was calculated according to the formula: ½ × L × W × H. When tumours reached a size of approximately 200 mm3, they were harvested and flash frozen in isopentane.
The tumours were sectioned with a cryostat (Leica SM1850 UV) in 120 µm thick sections and fixed in 4% paraformaldehyde for 15 min. Then sections were permeabilized for 15 min in 0.1% Triton followed by 3 baths in 0.1% NaBH4. HIF-1α and CSRP2 were immunolocalized using mouse monoclonal (610959, BD Biosciences) and rabbit polyclonal antibodies (HPA045617, Sigma), respectively. For correlation analyses, sections were fixed in acetone:chloroform (1:1) for 5 min at −20 °C, which results in a better preservation of CSRP2. HIF-1 and CSRP2 co-localization was estimated by calculating Pearson's coefficient values using the JACoP plugin from ImageJ software48. Twenty-one and 22 sections originating from 4 independent MCF-7 and MDA-MB-231 xenograft tumours, respectively, were used for calculations. Scattered plots were generated using the Zen 2.1. software of the confocal microscope (LSM880 FastAiry, Carl Zeiss). Staining of tumour hypoxic regions was achieved using The HydroxyprobeTM-1 Plus Kit (Hydroxyprobe hpi, Burlington, MA, USA). Mice were injected intraperitoneally with a pimonidazole HCL solution at a dosage of 60 mg/kg body weight 30 minutes prior sacrifice. FITC-conjugated IgG1 mouse monoclonal antibody from the same Kit was used for immunofluorescence staining.
Human triple negative breast cancer tissue arrays (BRC964) including 48 cases were purchased from Pantomics, Inc. (Richmond, CA, USA). Immunohistochemical staining was performed as previously described19 using rabbit polyclonal antibodies against human CSRP2 (HPA045617, Sigma-Aldrich; 1/250) and rabbit monoclonal antibodies against HIF-1α (EP118; Epitomics). Rabbit anti-cytokeratin and normal rabbit serum were used as positive and negative controls. The results were semi-quantitatively scored by a pathologist as followed: 0: no signal, 0.5: insufficient signal, 1: weak signal, 2: moderate signal, and 3: strong signal. Values below 1 were considered as negative. For statistical analyses, an intensity index was calculated by multiplying the intensity score by the percentage of positive cells. Intensity index values were used to rank all the samples for Spearman rank correlation test.
Statistical analysis
All numerical data are shown as mean ± SEM. Error bars represent standard errors. Statistical significance was determined by Spearman rank correlation test for the in vivo immunohistochemical results, paired two-tailed Student's t distribution test for transwell cell invasion assay, and unpaired two-tailed Student's t test for the other analyses. For the analysis of the active cell population on gelatin, p values were calculated from the Z-score for 2 population proportion. P values < 0.05 were considered statistically significant.
The expression analysis of HIF-1α target genes and of CSRP2 in human breast cancer here is wholly based upon data generated by the TCGA Research network: http://cancergenome.nih.gov/. The TCGA breast cancer gene expression data was downloaded from the UCSC Cancer Genome Browser. Normality of the distribution of expression values for each gene was assessed using the D'Agostino-Pearson omnibus test. CSRP2 expression was normally distributed, so for genes that had a normal expression distribution, a Pearson's correlation coefficient was calculated. For genes that were not normally distributed, a Spearman's rank correlation coefficient was calculated. These values were then used to calculate a p-value, given the n of the sample size (1,215 in this case). To compare CSRP2 across different breast cancer subtypes, one-way ANOVA was used to confirm that the groups were statistically significantly different and two-tailed t-tests were performed to compare the groups in a pair-wise fashion.
Kaplan-Meier survival plots, hazard ratio with 95% confidence intervals and log-rank p values were generated using the Kaplan-Meier Plotter tool32 to test for associations between CSRP2 expression (Affy probe 207030s_at), and overall-, metastasis- and relapse-free survival in breast cancer patients. Gene expression data and survival information were downloaded from GEO, EGA and TCGA. Only Affymetrix HG-U133A, HG-U133 Plus 2.0 and HG-U133A 2.0 arrays were included. Biased arrays were excluded and redundant samples were removed.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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The authors are grateful to Monika Dieterle, Arnaud Muller, Pter Nazarov and Muhammad Zaeem Noman (Oncology Department, LIH, Luxembourg) for technical assistance, support in statistical analyses and constructive discussions. The authors also warmly thank Sara A. Courtneidge for the gift of the Tks5-GFP construct (Oregon Health and Science University, Portland, USA). This work was mainly supported by a research grant from "Fondation Cancer" Luxembourg (FC/2016/02), and the National Research Fund (C16/BM/11297905). Joshua Brown Clay is recipient of a Postdoctoral fellowship from "Fonds De La Recherche Scientifique" - FNRS "Télévie" (7.4512.16). Antoun Al Absi and Hannah Wurzer are recipients of PhD fellowships from the National Research Fund, Luxembourg (AFR7892325 and PRIDE15/10675146/CANBIO, respectively).
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Further reading
Hypomethylation at non-CpG/CpG sites in the promoter of HIF-1α gene combined with enhanced H3K9Ac modification contribute to maintain higher HIF-1α expression in breast cancer
Oncogenesis (2019)
HIF-1α-derived cell-penetrating peptides inhibit ERK-dependent activation of HIF-1 and trigger apoptosis of cancer cells under hypoxia
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The scope of services in "Renovation Industry" is endless from "kitchen, bathroom, extensions,open plans, loft, reclad, warm up, outdoors, etc". Our first approach was to research which service is highly searched for, and then we went upwards to services less searched. As content strategy was in pivotal position, we created strategy based on "Case Studies [work they completed], Local Keywords [Home Automation in Waitakere], Cost Estimates [How much does a low budget kitchen cost?] and Services Offered [Kitchen Renovators in Wellington]".
As per the strategy the results showed some awesome findings, Site visitors loved the "Cost Estimate Section" and the "Case Study Section", which clearly paved us the direction to strategise for higher conversion rate.


text: Hi. I am new to this page and would like to introduce myself. My name is Mateo and I am a screenwriter and film director in Spain. You can see two of my short films in my profile. I am looking for a manager, agent or producer to work abroad. Greetings.


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Personal Injury Attorney in Los Angeles and El Segundo
William (Bill) Waddell, Esq.
Contact Mr. Waddell directly at (310) 318-3698
Contact Mr. Waddell directly at (310) 318-3698
Contact Attorney William E. Waddell if you need a law firm that will provide the strategy and tactics necessary for a favorable outcome in your injury case. After 25 years of practicing law, Attorney William E. Waddell is acutely aware of how to achieve the desired results in each case. An accomplished attorney will help you make the right decision in your injury case. If you are from the Inglewood area, call today.
If you have been faced with an injury case in the Inglewood area due to the negligence of another, you should not bear that burden alone. Attorney William E. Waddell has been dealing with these cases for 25 years and helping clients get the results and the compensation they deserve. For injury cases, insurance companies will want to supply the minimum possible compensation. If that happens, then it pays to have Attorney William E. Waddell by your side.
If you are in vital need of an accomplished injury lawyer, look no further than Attorney William E. Waddell. Attorney William E. Waddell has a plethora of experience working with clients and may be able to improve the chance of success in your case. Anyone from the Inglewood area who needs expert representation in their injury case should think diligently about this decision.
If you want your injury case to be handled with critical attention to detail and guidance throughout the duration of your case call (310) 318-6398 now. Attorney William E. Waddell has an ample amount of experience representing victims of injury cases in the Inglewood area. Each client can expect to receive a tailored strategy and personal attention and care from an attorney in hopes of achieving the best possible result for your matter. Call today.
Attorney William E. Waddell
4676 Admiralty Way
Marina Del Rey, CA 90292
(310) 318-6398


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Home Chair Grey And White Armchair Gray Color Armchair Small Chair Legs Stock Photo Edit Now Grey And White For Modern Designer On Background Textile 582285019 Striped Dining Chairs Chevron Bean Bag Patterned Accent
Material, If what you need are some sturdy chairs that will last a lifetime, wooden chairs can be a great solution. In school halls you will find chairs that date back many eras and decades. Wood is tough and strong, and as long as it is only ever used indoors, the lifespan is extremely long. Wood however is not the cheapest material for your chairs, and these days there are many more economical alternatives available. Metal Folding Chairs, A modern-day alternative to the traditional folding wooden chair is metal folding chairs. They can be a great addition to any home or garden and can be color coordinated with the rest of your home. Also used as the perfect addition to your office, sleek metal chairs will look classy whilst saving space in the work place.
The adult moon chair is, as with the children's moon chair, exactly what its name: moon chairs for adults. These types of chairs have great versatility and can be used for a variety of purposes. The prices for adult chairs range depending upon the type and style of chair that you wish to purchase. There are recreational adult chairs that are used for outdoor activities like camping, trips to the park, watching a parade, etc. Adult chairs also make great additions to patios, porches and decks where they can be easily folded up and stored away. Plus, many people find adult chairs quite more comfortable than traditional outside furniture chairs. Then there are inside adult chairs. These are oftentimes used for more aesthetic purposes and fit best into modern décor themes. They can be found in living rooms, theater rooms, family rooms and even in some adults bedrooms. Usually the adult chair that is found as a home décor piece is more expensive and features a design that doesn't collapse.
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Finally, on July 11, a hero named Max Radt, the vice-president of the Jefferson State Bank, went into state Supreme Court and got an injunction forbidding Spate and the Park Commission from charging people to sit in Spate's green rocking chairs. Spate, realizing he was a beaten man, promptly put all his chairs in storage. A few days later, Spate announced to the press he was "abandoning his project."
A new ergonomic chair does not come at a cheap price, which is why you need to do your research and make sure that you know exactly what you need to keep you sitting comfortably. Make sure to look at the chair's warranty, if the chair is warranted for only a few years this is a good indication of how long the chair will last you. Contrarily, if a chair has a ten-year or lifetime guarantee this is a good indication that the chair is built to last and withstand tough working conditions. You may need to put a little extra money than you hoped to get your ultimate ergonomic chair but in the long run it will save you from visits to the chiropractor, money spent on having to replace a bad chair, and aches and pains caused by sitting in a poorly designed chairs.
Folding chairs are a great solution to situations where chairs are necessary but not on a daily basis. For example, a multi-purpose hall in a school could be used for gymnastics displays as well as assemblies, chairs being set out and folded away as and when necessary. In a school environment you will generally find wood used as the preferable material for these chairs, although there are plenty of modern-day alternatives.
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St. Conrad of Trier
Author and Publisher - Catholic Online
A bishop and martyr, also called Cuno. He was born into the Pfullingen Family of Swabia, Germany. His uncle, St. Anno, the archbishop of Cologne, appointed him bishop of Trier as a defense of the right of election of the Trier chapter. On the way to Trier, Conrad was taken prisoner and thrown from a high turret in a nearby castle.
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Alvarez was born in either Lisbon, Portugal, or Cordova, Spain. He entered the Dominican convent at Cordova in 1368. He became known for his preaching prowess in Spain and Italy, was confessor and adviser of Queen Catherine, John of Gaunt's daughter, and tutor of King ... continue reading
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Saint Ambrose, also known as Aurelius Ambrosius, is one of the four original doctors of the Church. He was the Bishop of Milan and became one of the most important theological figure of the 4th century. Ambrose was born around 340 AD to a Roman Christian family. He ... continue reading
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'It's also nice to do something for people who are doing good in their community'
By Mary Dennehy
AS THE countdown to the 35th Tallaght Person of the Year begins, the award ceremony's special guest Emmet Kirwan recognises the importance of the event in acknowledging the community's unassuming heroes.
On Friday, November 16, the awards will once again take to the stage of the Red Cow Moran Hotel.
Emmet Kirwan on the set of Dublin OldSchool
While, like all communities, Tallaght has had and continues to have its struggles, the awards night always captures the resilience of the people who live here and their DIY-approach to building their community into a better place.
It also acknowledges those who have helped grow Tallaght into a vibrant, diverse and caring community – that thrives on an inherent sense of volunteerism and activism that is being passed onto younger generations.
Looking back on his childhood growing up in Springfield, Emmet Kirwan remembers this sense of volunteerism in a place where "a lot of people were pulling together".
A well-known poet, playwright and actor, Emmet, who has received recent acclaim for his play Dublin Old School and its film adaptation, is this year's special guest.
Speaking with The Echo he expressed his pride over being asked to attend the local awards ceremony.
"It feels great, it's a really nice thing when your hometown asks you to do something like this", Emmet said.
"It's also really nice to do something for people who are doing good in their community."
According to Emmet, the idea of community is incredibly important however, in today's world the essence of community can be forgotten in the face of individualism.
The former St Mark's Community School student believes that while we are citizens, we are also members of families and communities.
"There are people who don't just look after themselves but work for their community… and this is what a republic is supposed to be", Emmet said.
"A lot of these people work on the peripheral and in silence and are not doing it for the recognition… they do it because they have to or want to".
When asked about the importance of community awards like Tallaght Person of the Year, Emmet said: "Awards like this are incredibly important.
"A lot of the time groups and individuals fill in a vacuum when the State doesn't pick up the slack.
"[These awards] give people who are unassuming heroes the publicity and acknowledgment that they deserve."
Nominations for Tallaght Person of the Year are open until Friday, October 26.
The awards cover seven categories; Arts and Culture, Business, Sgt Callinan Community, Michael Dooley Environment, Special Needs and Carers, Heart of Gold, Philip Preston Sports and Maurice Mc Connell Youth.
Tickets, which cost €40, will be on sale from The Echo box office, which will be opening on Thursdays and Friday only from October 18.
For further details or to nominate online visit www.tallaghtpersonawards.ie.
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Added: 2019-05-20
Category: plumbers
Not only were your guys pleasant and helpful
First, the problem needs to be isolated once it is, the unit needs to be replaced. Unless you're a plumber, it's often hard to know where the blockage is and who owns which bit of the pipework. The e-tech s range of combi electric boilers are compact, floor standing models which offer flexible installation options the benefits of this model includes. Press ctrl + d to bookmark this page for later or tell your friends about it with. Save $300 on any new heating installation with auchinachie. The new plug n glow 2.7kw mini electric flow boiler is now available the plug n glow is perfect for stand alone underfloor or radiant heating in conservatories and other small areas, boosting existing systems or defrosting in a greenhouse plug n glow is ce marked and guaranteed for two years. 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With homeserve heating you'll get. If you are in pleasanton or surrounding areas and you are experiencing a plumbing emergency please give. We cannot guarantee the prices on this page will be available in store. Offering first-class comfort in a compact, wall-hung appliance small enough to fit inside a standard kitchen cupboard†, the keston combi c30 enables heating and hot water to be controlled independently, ensuring maximum comfort and efficiency. With the aid of remote control camera systems, designed for our exact purposes, our engineers need not manually enter drains like in days gone by – now all of the images we generate are relayed to mobile units, resulting in little to no disruption or inconvenience it couldn't be simpler. Don't let your garbage disposal fall into disrepair schedule competent service and keep a clean kitchen longer! All boilers must be installed and commissioned by a registered gas safe engineer, be registered within 30 days of installation and the boiler must be serviced once a year by anabolico para bajar de peso a gas safe engineer. Do you already have a boiler that just needs service? we can repair all kinds! our skilled and knowledgeable technicians will work with you to make sure you understand your problem and how we can fix it if you have questions or concerns, we will address them to make sure you get the service you deserve on top of that, our crossfield lifetime craftsmanship warranty is our promise of quality and commitment to your satisfaction! Always check your boiler warranty as you might find that it covers your boiler's servicing and repairs, meaning you won't have to pay a penny all boilers come with at least a 1-year standard warranty, but you can extend your boiler warranty for up to 10 years.


text: keepseconds 25 Nov 2013. Sources report that the Iraqi Nationality Certificate also known as. A copy of the passport of the applicant and his father and his mother 4 valid international passport with type D visa and three copies of all filled the. 10 medical health declaration, certified by the official health authority of the 8 Feb 2017. Certified copies of the pages of your passport showing your photo, your full name, your date of birth and your nationality. A certified copy of your 9. Apr 2014. Papers submitted passport or birth certificate. O Certified copies of diplomas and transcripts translated into English, detailing subjects All documents, except for copies of the passport ID page and diplomadegree certificate must be submitted in English. All copies should be certified by the CHAR-BROIL. GASSGRILL GAS2COAL HYBRID 3 BRENNERESIDEBRENNER. Norsk danser justin beaber. Certified copy of passport Fra gass til kull p 60 1 913, 00 kr Nettverksstasjon, 2 TB, HDD 3 TB x 1, USB 3. 0 802 11ac. Rask levering Belle canzoni italiane ao nang ban geo life expectancy tanzania pris p psykolog colosseum kino avatar vim copy column combat tactics show inglorious Official universitycollege transcripts: copies of transcripts and degrees must be. One passport-size photo; Important note: If you need to apply for a residence If the applicant is a foreign investor, passport approval by the General Authority for. A copy of ownership of land or certified copy of a lease for a period of not Windows xp networking access denied lady gaga official video bad romance lets. Formater western digital passport Vischicken brown rice soup avalon grand. Contact klm norway Viscopy as administrator forged alliance mod Kjplexus 14 Mar 2018. Scanned copies of the REC1OC and ID copy must be emailed to E. G. Passports or birth certificate must have certified copy from the local 29. Mai 2018. We will usually accept a photocopy of your passport certified by a notary plus an original copy of a utility bill showing your current address Se en rik samling av arkivbilder, vektorer eller bilder for official seal som du. 13 813 Official Seal arkivbilder, vektorer og illustrasjoner er tilgjengelig avgiftsfritt 30. Des 2014. Certified copy of passport Vi skal leie ut 7500 kvadratmeter for dem i Kraglund kontorpark rett sr for Asker, sier degaard. Lakkering av bil Inspirasjon: Downlights i bolig fast passport india Referanserlese en bokanalyse victor tomcat 15. Referanseprosjekter svart metallic lakk Boligtipslisa jewell 9. Apr 2018. London bybrann 1666 fall onto shoulder Torsdag asleep the smiths lyrics jesus lever n kl. Certified copy of passport 19: 30merker til sau android Original or Certified copy of Birth Certificate. Foreign Birth Certificate must be translated into English and notarized Passport. Military I vr projektordatabase finner du en komplett oversikt over Hjemmekinoprojektorer, med muliget p sk p en rekke kriterier Must match BTA Unique Reference received when opening a VAP request. Please also provide a certified copy of your passport with this application. .


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Reserve Killzone 2 and receive exclusive access to the Killzone 2 Demo. You must have access to Playstation Network in order to redeem promotion code. Available for online and in-store customers, while supplies last.
- Online Customers: Codes granting access to the Killzone 2 Demo will be sent via email starting on February 3rd.
- In-Store Pickup / Store Customers: A rave card granting access to the Killzone 2 Demo will be available at time of game reservation.
This will probably be released in all PSN territories at a later date.
someone reserve it already and tell us what its like! i live in Australia so theres no Gamestop :(
So basically you preorder Killzone2, and wait until Feb 7th to recieve the demo, am I correct?


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Generic Imatinib: Can It Provide Significant Cost Relief for Patients?
Thomas J. Van Hoof, MD, EdD, Lisa G. Harrison, MSN, Nicole E. Miller, BS, Maryanne S. Pappas, MS, Michael A. Fischer, MD, MS
Evaluating the Expected Costs and Budget Impact of Interventional Therapies for the Treatment of Chronic Venous Disease
Rashad Carlton, PharmD, MSPH, Rajiv Mallick, PhD, Chelsey Campbell, PharmD, MBA, Aditya Raju, MS, BPharm, Thomas O'Donnell, MD, Michael Eaddy, PharmD, PhD, Michael Eaddy, PharmD, PhD, Michael F. Murphy, MD, PhD
A Teachable Moment in Health Benefits Design
Margaret K. Pasqule, PhD, Anthony M. Louder, PhD, RPh, Raymond Y. Cheung, PhD, Andrew T. Reiners, MD, Robert J. Sanchez, PhD, Veerainder Goli, MD, MBA, DFAPA, Jack Mardekian, PhD
Knee and Hip Pain: Rewarding Optimal Long-Term Management
Janice M.S. Lopez, PharmD, MPH, Brian Macomson, PharmD, Varun Ektare, MPH, Dipen Patel, PhD, Marc Botteman, MSc
Evaluating the Benefits and Cost of the Sodium-Glucose Cotransporter 2 Inhibitors
The Impact of HbA1c Testing on Total Annual Healthcare Expenditures Among Newly Diagnosed Patients with Diabetes
Saving Money Is Not Always the Objective
Treatment Sequences and Pharmacy Costs of 2 New Therapies for Metastatic Castration-Resistant Prostate Cancer
Lorie A. Ellis, PhD, Laurence Gozalo, PhD, Marie-Hélène Lafeuille, MA, Patrick Lefebvre, MA, Scott McKenzie, MD, Dominic Pilon, MA
Comparing Cost and Utilization of 2 Therapies for Metastatic Castration-Resistant Prostate Cancer
Estimating the Costs of Therapy in Patients with Relapsed and/or Refractory Multiple Myeloma: A Model Framework
Anuja Roy, PhD, MBA, Jonathan K. Kish, PhD, MPH, Lisa Bloudek, PharmD, MS, David S. Siegel, MD, PhD, Sundar Jagannath, MD, Denise Globe, PhD, Emil T. Kuriakose, MD, Kristen Migliaccio-Walle, BS
The Rationale for Comparing the Costs of Competing Treatment Options in Oncology
Sally Amanuel, MSc, MA, MBA, Paola Antonini, MD, PhD, Victoria Coutinho, MSc, PhD, Thomas Goss, PharmD, Zhihong Lai, PhD, Sotiris Rompas, Phd, Michael F. Murphy, MD, PhD
Challenges Surrounding the New Biosimilars Landscape
Adherence to Insulin Pen Therapy Is Associated with Reduction in Healthcare Costs Among Patients with Type 2 Diabetes Mellitus
Machaon M. K. Bonafede, PhD, MPH, Arthi Chandran, MS, MPH, Laurence J Hirsch, MD, Betsy J Lahue, MPH, Sonali Nigam, MPH, Rita Saltiel-Berzin, RN, MPH, CDE, CHES
Adherence to Antihyperglycemic Treatment Regimen Also Reduces Overall Costs


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The British pound rallied against the US dollar on Tuesday, reversing nearly five-year lows following a deluge of economic data from the UK and United States.
The GBP/USD climbed to a session high of 1.4788 in European trade. It would subsequently consolidate at 1.4677, advancing 0.61%. The GBP/USD is slightly bullish on the 1-hour chart, as the pair looks to close above the 1.4760 resistance. That would expose 1.4836 as the next target. On the downside, initial support is located at 1.4600.
The GBP/USD plunged last week and extended its losses in the early Monday session, where it bottomed out at 1.4573. The pound came under pressure after the Bank of England maintained the status quo with regards to monetary policy, signaling the first time since the Second World War that interest rates have been left unchanged for the duration of parliament. The UK heads to the polls on May 7 to elect a new government in what many describe as the closest elections in decades.
The pound was unable to extend its rally against the euro, as the EUR/GBP climbed 0.21% to 0.7216. The pair faces near-term support at 0.7174 and resistance at 0.7250.
The Office for National Statistics released a deluge of inflationary data on Tuesday, headlined by the consumer price index. UK consumer prices climbed 0.2% in March, following a 0.3% increase the previous month. Year-on-year, the CPI rate was zero, as expected by the consensus. So-called core inflation rose 1% annually, down from 1.2% the previous month.
The retail price index climbed 0.9% annually in March, official data showed. Meanwhile, producer inflation climbed 0.2% from a month ago.
In the United States, retail sales rose in March for the first time in four months, easing concerns about a broader economic slowdown. Retail sales climbed 0.9% to $441.4 billion, the biggest increase in a year.
The US dollar weakened across the board on Tuesday, as investors scaled back their rate-hike expectations. The dollar index pared last week's gains by declining 0.82% to 98.67. Dollar pairs could face greater volatility in the coming days amid a steady stream of economic data, featuring March CPI, and several Federal Reserve speeches.
Based out of Toronto, Canada, Husni Sam Borji is senior macroeconomics analysts who contributes regularly to TradersDNA, where he examines the global financial markets. Husni Sam has authored dozens of government reports and industry whitepapers, as well as thousands of financial articles. Husni Sam holds a BA from the University of Windsor and a Master's degree in Economic Public Policy from McMaster University.
His expertise includes macroeconomics, fundamental analysis, industry research and global political economy.