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Melanoma Chemotherapy Leads to the Selection of ABCB5-Expressing Cells
Marine Chartrain ,
Contributed equally to this work with: Marine Chartrain, Joëlle Riond
Affiliations UMR 2587, CNRS-Pierre Fabre, Institut des Sciences et Technologies du Médicament de Toulouse (ISTMT), Toulouse, France, Centre de Recherche en Oncologie Expérimentale, Centre de Recherche et Développement Pierre Fabre - Toulouse Langlade, Toulouse, France
Joëlle Riond ,
* E-mail: joelle.riond@dr14.cnrs.fr
Affiliations UMR 2587, CNRS-Pierre Fabre, Institut des Sciences et Technologies du Médicament de Toulouse (ISTMT), Toulouse, France, USR 3388, CNRS-Pierre Fabre, Centre de Recherche et Développement Pierre Fabre - Toulouse Langlade, Toulouse, France
Aline Stennevin,
Affiliation Centre de Recherche en Oncologie Expérimentale, Centre de Recherche et Développement Pierre Fabre - Toulouse Langlade, Toulouse, France
Isabelle Vandenberghe,
Bruno Gomes,
Laurence Lamant,
Affiliation INSERM U563, CHU Purpan, Toulouse, France
Nicolas Meyer,
Affiliation Hôpital Larrey, Toulouse, France
Jean Edouard Gairin,
Affiliations UMR 2587, CNRS-Pierre Fabre, Institut des Sciences et Technologies du Médicament de Toulouse (ISTMT), Toulouse, France, UMR152 IRD-UPS "PHARMA-DEV", Faculté de Pharmacie - Université Toulouse III - Paul Sabatier, Toulouse, France
Nicolas Guilbaud,
Jean Philippe Annereau
Marine Chartrain,
Joëlle Riond,
Marine Chartrain Joëlle Riond ... Jean Philippe Annereau
Metastatic melanoma is the most aggressive skin cancer. Recently, phenotypically distinct subpopulations of tumor cells were identified. Among them, ABCB5-expressing cells were proposed to display an enhanced tumorigenicity with stem cell-like properties. In addition, ABCB5+ cells are thought to participate to chemoresistance through a potential efflux function of ABCB5. Nevertheless, the fate of these cells upon drugs that are used in melanoma chemotherapy remains to be clarified. Here we explored the effect of anti-melanoma treatments on the ABCB5-expressing cells. Using a melanoma xenograft model (WM266-4), we observed in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment that induces a significant tumor regression. These results were further confirmed in a preliminary study conducted on clinical samples from patients that received dacarbazine. In vitro, we showed that ABCB5-expressing cells selectively survive when exposed to dacarbazine, the reference treatment of metastatic melanoma, but also to vemurafenib, a new inhibitor of the mutated kinase V600E BRAF and other various chemotherapeutic drugs. Our results show that anti-melanoma chemotherapy might participate to the chemoresistance acquisition by selecting tumor cell subpopulations expressing ABCB5. This is of particular importance in understanding the relapses observed after anti-melanoma treatments and reinforces the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma.
Citation: Chartrain M, Riond J, Stennevin A, Vandenberghe I, Gomes B, Lamant L, et al. (2012) Melanoma Chemotherapy Leads to the Selection of ABCB5-Expressing Cells. PLoS ONE 7(5): e36762. https://doi.org/10.1371/journal.pone.0036762
Editor: Meenhard Herlyn, Wistar Institute Program, United States of America
Received: June 29, 2011; Accepted: April 12, 2012; Published: May 24, 2012
Copyright: © 2012 Chartrain et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Support for equipment was provided by the Fonds Européen de Développement Economique Régional which had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Melanoma is one of the most aggressive form of skin cancer and its incidence is increasing worldwide, especially where fair-skinned people receive excessive sun exposure [1]–[3]. Primary tumors without any evidence of metastases are mostly treated by surgery. However, metastatic melanoma is highly resistant to conventional radio and chemotherapies and remains a disease of poor prognosis, with median survival times comprised between 7 and 9 months. A number of chemotherapeutic agents (such as dacarbazine, temozolomide or fotemustine) alone or in combination have a limited activity with relatively low response rates (<25% for any single agent) [4], [5] and so far only a small impact on overall survival. Even if some promising targeted therapies are currently developed with new BRAF kinase inhibitors such as vemurafenib [6], melanomas invariably become resistant to these agents [7]. Thus, chemoresistance remains a serious concern for melanoma therapy.
Besides the mechanisms of resistance to chemotherapy that are shared by various cancers, melanomas display specific features. Melanoma cells are equipped with melanogenesis-related vesicles, the melanosomes, that have been shown to be involved in drug trapping and export [8]. Secondly, these cells express ABC transporters which have been associated with multidrug resistance by lowering the intracellular accumulation of cytotoxic drugs [9]. Of particular interest is ABCB5 which shares 73% of sequence homology with ABCB1 (P-gp, MDR1) [10], [11]. Firstly detected in tissues derived from the neuroectodermal lineage including melanocyte progenitors [10], melanoma cell lines and patient specimens [11]–[14], ABCB5 expression was also found in other tissues [15]–[17] but is restricted to a subpopulation of cells. In melanoma, ABCB5-expressing cells are endowed with self-renewal, differentiation and tumorigenicity abilities [18], [19]. Their abundance in clinical melanoma specimens correlates positively with the neoplasic progression suggesting that ABCB5 expression is associated with tumor aggressiveness. Moreover, the growth of melanoma xenografts in mice was delayed when the animals were treated with a monoclonal anti-ABCB5 antibody [18].
As a member of the ABC transporter family, ABCB5 is thought to play a role in drug efflux. This was supported by experiments measuring the intracellular accumulation of Rhodamine 123 [10] or doxorubicin in melanoma [14] and hepatocarcinoma cells [15], [16]. Nevertheless, the level of resistance of melanoma ABCB5-expressing cells to relevant chemotherapeutic drugs remains unknown. Here, we demonstrated that ABCB5+ cells display a survival advantage over ABCB5− cells upon anti-melanoma treatment. We show that: 1) ABCB5-expressing cells selectively survive over ABCB5− cells after a temozolomide treatment inducing a significant tumor regression in the WM-266-4 xenograft model 2) ABCB5-expressing cells are more abundant in melanomas from patients treated with dacarbazine 3) in vitro, dacarbazine but also vemurafenib and other drugs induce an increase in the ABCB5-expressing cell population at doses that are cytotoxic for the bulk cells.
ABCB5 Expression is Restricted to a Subpopulation of Melanoma Cells
To assay the variation of the expression of ABCB5, we first devised the methodology to detect the presence of ABCB5-expressing cells in a panel of melanoma cell lines using a rabbit polyclonal antibody raised against a peptide derived from the ABCB5 protein sequence (ABCB5-AbRock). In the WM-266-4 cell line, this antibody consistently labels a subpopulation of living cells that can be distinguished among the bulk of cells by flow cytometry (figure 1A). The proportion of positive cells is low but significant and reproducible within the repeated experiments. The specificity of the labelling was assessed by depletion experiments using RNAi interference. The siRNA-mediated down-regulation of the ABCB5 mRNA expression, as measured by Q-PCR, was associated with a strong reduction of the ABCB5+ population to 37% of the control (figure 1B). In addition, we confirmed that ABCB5-expressing cells detected with the ABCB5-AbRock antibody are also labelled by the monoclonal 3C2-1D12 one (ABCB5-AbAbD) described earlier [10], [14] (data not shown).
Figure 1. ABCB5 is expressed on the surface of a subpopulation of melanoma cells.
WM-266-4 cells were surface-labelled with the ABCB5-AbRock antibody and analyzed by flow cytometry. ABCB5+ cells (right contour plot) were gated on viable cells (DAPI-negative) according to the isotype control (left contour plot). Inserts (dot plots) display the gating of the positive cells (A). WM-266-4 cells were treated with a siRNA designed to target ABCB5 (si-ABCB5). After 72 h, the cells were analyzed for their ABCB5 mRNA content and ABCB5 surface expression. The left and right histograms show respectively the relative expression of ABCB5 mRNA normalized to the ABCB5 mRNA in cells treated with a control siRNA (si-ctrl), and the percentage of ABCB5+ cells among total cells (n = 3). The corresponding contour plots are shown (B). Different melanoma cell lines were analyzed for their ABCB5 surface expression (C) or their ABCB5 mRNA content (D) (n = 3).
The presence of such an ABCB5+ subpopulation is not restricted to the WM-266-4 cell line model and was detected in most of the tested melanoma cell lines, with a proportion ranging from 1 to 5% of the total cells (figure 1C). Interestingly, the amounts of the ABCB5 mRNA quantified by Q-PCR and the percentages of the ABCB5-expressing cells appear to be correlated for four of the five tested cell lines (figure 1C and D), with the exception of G361 cells in which an ABCB5+ subpopulation was detected despite a relatively low abundance of mRNA.
Of note, the WM-115 cell line derived from a primary melanoma tumor expresses six times less ABCB5 mRNA than the WM-266-4 cell line which originates from a metastasis of the same patient.
ABCB5-expressing Cells are Enriched in the Residual Tumors after an Anti-melanoma Treatment in vivo
We investigated the effect of an anti-melanoma chemotherapeutic treatment on the ABCB5-expressing cell subpopulation in vivo. This was assayed using WM-266-4 tumor cells subcutaneously xenografted in nude mice. Temozolomide was selected as the anti-melanoma substance. Mice bearing 14-days tumors were treated at days 14, 16, 18, 21 with the drug doses known to induce a significant anti-tumor effect. Tumor growth was followed over a 21 days period (figure 2A) and tumor specimens were harvested and analyzed for their content in ABCB5-expressing cells at days 17 and 22. As shown on figure 2B, an increase in the ABCB5+ cells ratio is detected 24 h after the second injection of temozolomide, concomitantly with an efficient tumor growth inhibition. This ratio increases again at day 22 after two more drug administrations when a severe mass reduction is observed. Immunohistochemical analysis of mice tumors with a different antibody confirms an increased expression of ABCB5 in tumors from the treated mice (figure 2C).
Figure 2. ABCB5-expressing cells are enriched in the residual tumors after an anti-melanoma treatment in vivo.
WM-266-4 cells (5×106 cells) were injected subcutaneously in Swiss nude mice. Fourteen days later, mice were treated by repeated i.p. injections of either temozolomide (80 mg/kg) or vehicle following the schedule indicated by the black arrows. The tumoral volumes were monitored and the means of measured volumes respectively for temozolomide-treated and vehicle treated tumors are as follows: 180 mm3 and 175 mm3 at day 14; 264 mm3 and 295 mm3 at day 16; 178 mm3 and 444 mm3 at day 18; 84 mm3 and 699 mm3 at day 21. (A). 24 h after the injections at days 16 and 21 (d17 and d22), tumors were recovered, dissociated and the cell suspensions were searched for the presence of human ABCB5+ cells by flow cytometry (B) (medians are represented as black lines). Tumors recovered at day 17 were analyzed by immunohistochemistry for their ABCB5 expression (C).
ABCB5 Expression is Enhanced after Treatment in Clinical Samples
We analyzed the expression of ABCB5 in human melanoma metastatic samples obtained from unmatched patients, before and after treatment. The figure 3 shows that 7 of the 8 samples from untreated patients display low-staining intensity (on a level-scale based on the extent and intensity of the staining on the whole tumor section). By contrast, 4 of the 7 tumors from treated patients display a higher level of ABCB5 staining. While this difference is not statistically significant due to the low number of examined samples, it nevertheless suggests a higher expression of ABCB5 in tumors from patients having received treatment.
Figure 3. ABCB5 expression is increased in melanoma tumors obtained from treated patients.
Skin metastases specimens from respectively 8 untreated and 7 treated patients were analyzed by immunohistochemistry for their ABCB5 protein expression. The ABCB5 staining intensity was ranked in four arbitrary classes according to the intensity and the extent of the labelling. Representative staining of two levels of intensity (left panel: isotypic control) (A). Repartition of the specimens in the different classes (B). The two groups of specimens (untreated versus treated) have been compared with the non parametric Kruskall Wallis test (p<0.30).
ABCB5-expressing Cells Enrichment upon Cytotoxic Treatment is Due to their Selective Survival
We aimed at understanding the origin of the ABCB5-expressing cells enrichment we observed in vivo. WM-266-4 cells were treated in vitro with dacarbazine which is structurally related to temozolomide and is active in vitro. Total and ABCB5+ cells numbers were monitored simultaneously after a 72 h treatment, using a range of cytotoxic drug concentrations. While the number of the bulk cells decreases with increasing concentrations of drug, the number of ABCB5+ cells remains above their basal number in untreated sample (figure 4A). The survival advantage of ABCB5+ cells was also found in G-361 and SK-MEL-28 cell lines (figure 4B and 4C) and was confirmed with a different antibody (data not shown). In addition to the survival, an increase in ABCB5+ cell numbers were detected to a certain extent in the different cell lines analyzed. This was particularly clear in the WM-266-4 cells in which ABCB5+ cells are increased by a two-fold factor (figure 4A). The dose-response profile is biphasic, with a maximum effect at a concentration that induces 50–75% of cytotoxicity on the bulk cells. At higher concentrations, the ABCB5+ cells numbers decrease and reach the basal control value (100%). The same response profiles were obtained when melanoma cells were treated with vemurafenib (figure 4D–4F). Moreover, the numbers of ABCB5+ cells detected upon vemurafenib are significantly higher than with dacarbazine. By contrast, under doxorubicin exposure, the numbers of ABCB5+ cells decrease with increasing concentrations suggesting that they are sensitive to the cytotoxic treatment. Nevertheless, they display a slight survival advantage over the bulk cells (figure 4G–4I).
Figure 4. ABCB5-expressing cells survive upon dacarbazine treatment.
WM-266-4 (A,D,G), G-361 (B,E,H) and SK-MEL-28 cells (C,F,I) were treated at the indicated concentrations of dacarbazine (A–C), vemurafenib (D–F) and doxorubicin (G–I). After 72 h, the total viable cells were numbered using an automated cell viability analyzer. The percentages of viable ABCB5-expressing cells (among viable cells gated on DAPI-negative cells) were analyzed by flow cytometry. The numbers of total cells (white symbols) are reported as percentages of the number of cells in the untreated control sample. The numbers of viable ABCB5+ cells (black symbols) were calculated from the total cell numbers and ABCB5+ cells ratio, and reported as percentages of the viable ABCB5+ cells number in the control sample. ABCB5+ cells represent respectively 3%, 3.5% and 5% of the total cells in the WM-266-4, G-361 and SK-MEL-28 cell lines.
Dacarbazine Treatment Stimulates the Neo-synthesis of the ABCB5 Protein and its Exposure at the Cell Surface
The results shown above suggest that, in addition to being less cytotoxic for ABCB5+ cells than for the bulk cells, dacarbazine has other effects that participate to increase the ABCB5+ cells number detected after treatment. In order to determine a possible role of protein neo-synthesis and vesicular trafficking, we studied the effect of cycloheximide and brefeldin A on ABCB5 expression. We measured both the level of ABCB5 at the cell surface via the fluorescence intensity and the number of cells expressing a detectable level of ABCB5 via the percentage of ABCB5+ cells (figure 5 and figure S1). Cycloheximide does not affect the basal level of ABCB5 at the surface of untreated cells, but reverses its increase when cells are treated with either dacarbazine or doxorubicin (figure 5A). Concomitantly, it reduces the percentage of ABCB5+ cells in untreated cells or in dacarbazine-treated cells. Cycloheximide has no significant impact on the number of ABCB5+ cells after doxorubicin treatment (figure 5B).
Figure 5. Enrichment in ABCB5-expressing cells is associated with protein neo-synthesis and ABCB5 relocation at the cell-surface.
WM-266-4 cells were treated with dacarbazine, doxorubicin or vehicle (NT) for 72 h. Cycloheximide (A–B) or brefeldin A (C–D) were added respectively 24 h and 4 h before the treatment end-point. Cells were labelled for ABCB5 and analyzed by flow cytometry. The means of fluorescence intensity of the ABCB5+ cells from three independent experiments were reported in A and C. Fluorescence intensity histograms of representative experiments are shown in the figure S1. The number of ABCB5+ cells was reported in B and D as a percentage of the ABCB5+ cell number in the vehicle-treated sample.
Brefeldin A has no effect on either the basal expression of ABCB5 or the basal number of detected ABCB5+ cells in untreated cells. But it reduces both parameters in dacarbazine-treated cells suggesting that trafficking participates to increase the ABCB5 expression at the cell surface. No significant effect was seen with doxorubicin.
Selective ABCB5-expressing Cells Survival upon Dacarbazine Occurs with Other Clinically Used Drugs and is Unrelated to ABCB1 Expression
The ABCB5 protein is barely detectable in untreated WM-266-4 cells by Western blot analysis (figure 6A). By contrast, treatment with dacarbazine, vemurafenib or gemcitabine (which also leads to enrichment of ABCB5-expressing cells) induces an increased signal corresponding to a protein at the expected molecular weight of 85 kDa. Concordantly with the lack of increase of ABCB5+ cells under doxorubicin exposure, no enrichment of the ABCB5 protein was seen in doxorubicin-treated samples.
Figure 6. Quantification of ABCB5- and ABCB1-expressing cells after cytotoxic treatments.
WM-266-4 cells were treated for 72 h with the indicated concentrations of doxorubicin, dacarbazine, vemurafenib, gemcitabine or with vehicle (NT) and ABCB5 expression was analyzed by Western blot. Band intensities were quantified and variations are indicated as fold increases in treated versus untreated samples (A). WM-266-4 cells were treated with various drugs at their EC50 for 72 h. The percentages of positive cells among surviving cells were measured by cell surface labelling and flow cytometry analysis for ABCB5 (B) or ABCB1 (C). The relative mRNA expression of ABCB5, ABCB1, ABCC1, ABCG2 and HMBS as the house-keeping gene was measured by Q-PCR (see also table S1) and the amplified products were run on agarose gel after 29 cycles except for ABCB1 (32 cycles) (D).
We analyzed the effect of different other drugs on ABCB5-expressing cells. The percentages of ABCB5+ cells were monitored by flow cytometry after 72 h-treatments at their respective EC50 (figure 6B). All the tested drugs except doxorubicin increase by a factor 3 to 9 the percentage of ABCB5+ cells among the surviving cells.
Because the expression of different ABC transporters has been observed in melanoma, we evaluated their potential implication in the ABCB5+ cells survival upon dacarbazine as well as other chemotherapeutic agents. As shown by flow cytometry experiments (figure 6C), ABCB1 is expressed on a very rare subpopulation in untreated cells. By contrast with the ABCB5-expressing cells, the ABCB1+ subpopulation is not enriched after a dacarbazine (or gemcitabine) treatment. Non significant increases in the percentages of ABCB1+ cells were found after treatment with vindesine, paclitaxel, camptothecin and etoposide. Q-PCR experiments confirmed the low level of ABCB1 expression in the WM-266-4 cells compared to the ABCB1+ cells KB and KB-V1 (figure 6D and table S1). Some variations were detected in the ABCB1 expression after drug treatment, but they are not statistically significant.
The expression of ABCC1 and ABCG2 was also measured by Q-PCR. While these transporters are more abundant than ABCB1, their expressions remain unchanged after drugs treatments (figure 6D and table S1).
Reference chemotherapies used in clinics demonstrated their impact on acquired resistance of tumor cells. The resistance phenotype can then be exploited by the tumor cells as a selective advantage to reconstitute the tumor mass [20]. In the present work focused on melanoma, we show that ABCB5+ cells have a survival advantage over the bulk of tumor cells when they are exposed to different cytotoxic compounds, including drugs that are used in melanoma treatment.
ABCB5 expression was initially detected in a subpopulation of a melanoma cell line (G3361), as well as in melanoma samples obtained from patients [10], [18]. Here, we re-examined ABCB5 cell-surface expression by flow cytometry in five melanoma cell lines using a different anti-ABCB5 antibody [21]. As previously shown elsewhere [14], [18], we noticed that a subpopulation of cells was consistently surface-labelled. We confirmed the specificity of this labelling by ABCB5 silencing experiments and co-labelling experiments with a different anti-ABCB5 antibody. The proportions of this ABCB5+ cells subset vary in the different cell lines but stay in a range that is similar to the frequency reported for the G3361 cell line [14]. This variability is in agreement with previous analyses of a panel of melanoma cell lines [13], [14], [21] and was confirmed when measuring the ABCB5 mRNA content by Q-PCR.
When examining the ABCB5 mRNA expressing cell lines, we found a significantly higher proportion of ABCB5+ cells in the WM-266-4 cell line that originates from a metastasis, compared to the WM-115 cells from the primary tumor of the same patient. Interestingly, it was previously shown that ABCB5 expression level is correlated with the grade of melanoma [18]. Our results suggest that the couple WM-115/WM-266-4 tumor cell lines might be an interesting in vitro model to study the role of the ABCB5 protein and of ABCB5-expressing cells in melanoma aggressiveness.
The numbers of ABCB5-expressing cells detected by flow cytometry correlate with the ABCB5 mRNA level except in the G-361 cell line. In WM-266-4 cells, we showed that inhibitors such as brefeldin A and cycloheximide affect this number. This suggests that, in addition to RNA level, expression of ABCB5 at the cell surface depends also on the rate of the protein synthesis and trafficking toward the cytoplasmic membrane. We thus can not exclude that some of these events are particularly enhanced in the G-361 cell line, leading to the detection of a significant number of ABCB5-expressing cells despite a low abundance of ABCB5 mRNA as measured on the total cell population.
As a member of the ABC transporters family, ABCB5 was suggested to participate to the chemoresistant phenotype of melanoma cells [1], [12], [14]. A direct role of ABCB5 as a functional ABC transporter was assayed with doxorubicin, using the fluorescent properties of this well known ABCB1 substrate [14], [16], [21]. Nevertheless the behaviour of the ABCB5+ subpopulation upon anti-melanoma drugs remains unknown. We addressed this question taking advantage of the anti-melanoma effect of temozolomide on subcutaneaous WM-266-4 tumors grafted in mice. Temozolomide is a pro-drug that, once metabolized, acts with a similar molecular mechanism and efficiency than dacarbazine [22]. Its therapeutic potency is well mimicked in our xenograft model since an objective tumor regression was measured. Here we show that, in vivo, the regression of melanoma tumor upon temozolomide treatment is associated with a selective survival and an increased number of cells that express ABCB5 protein on their surface.
In order to extend this observation to human patient tumors, we analyzed the ABCB5 expression in clinical samples. Since ABCB5 expression has been shown to vary according to the melanoma stage [18], we restricted our study to skin metastases from untreated patients and from patients who relapsed after a first chemotherapy episode. Interestingly, the highest levels of ABCB5 expression were found in metastases from treated patients. This result is consistent with our in vivo findings and provides additional support to the observation that ABCB5-expressing cells are enriched upon anti-melanoma treatment.
We investigated this apparent ABCB5+ cells enrichment in vitro by monitoring the absolute number of ABCB5-expressing cells. We focused our analysis on the effects of dacarbazine that was, until now, the reference treatment for metastatic melanoma [4] and vemurafenib that recently proved efficiency in melanoma with BRAF V600E mutation [6]. Using different cell lines that bear the BRAF V600E mutation, we showed that ABCB5+ cells survive to both drugs at doses that are efficient in killing ABCB5− cells. The loss of viability detected at higher doses suggests that the mechanisms triggered to protect the cells from the cytotoxic stress are overwhelmed and, consequently, become inefficient. By contrast, ABCB5+ cells are more sensitive to the doxorubicin cytotoxic effect. In vitro cell treatments with dacarbazine were performed as long as two weeks (not shown). During the first week of treatment, we observed that the proportion of ABCB5-expressing cells continues to increase while the total number of cells decreases. After two weeks of treatment without interruption, proliferation resumes. Simultaneously, the proportion of ABCB5+ cells progressively decreases to a percentage that is similar to the percentage found in untreated cells. These results suggest the long-term surviving of cells and the recurrence of a heterogeneous population after a chemotherapeutic episode.
Both dacarbazine and doxorubicin stimulate the up-regulation of ABCB5 at the cell surface through protein neo-synthesis and trafficking. This might explain the slight difference consistently found between ABCB5+ cells and bulk cells cytotoxicity curves obtained with doxorubicin. But, as previously reported [23], we did not detect a doxorubicin-induced up-regulation by Western blot analysis since the frequency of ABCB5+ cells remains unchanged and stays very low after the treatment. Interestingly, dacarbazine has an additional effect leading to the neo-expression of ABCB5 at the surface of some cells. As previously reported with other substances [11], it up-regulates the ABCB5 mRNA level, but interacts also with the protein neo-synthesis. In addition, it induces the surface expression of ABCB5 on some cells through a process related to the intracellular vesicular traffic. The mechanisms underlying this observation remain to be understood.
We extended our analysis to a panel of cytotoxic compounds and found that drugs from various therapeutic classes induce a substantial increase in ABCB5+ cells in the surviving cellular population. One of the most potent chemoinducer is camptothecin. Interestingly, ABCB5 gene expression was found inversely correlated to camptothecin activity among cell lines of the NCI panel [14]. Furthermore, depletion using a siRNA that targets a short variant of ABCB5 was shown to sensitize melanoma cells to the camptothecin cytotoxic effect [12].
The peculiar cytotoxicity of doxorubicin on ABCB5-expressing cells remains to be understood. It suggests that the efficiency of ABCB5 as a transporter might be higher for drugs such as dacarbazine, vemurafenib, camptothecin, gemcitabine than for doxorubicin. To date, no transfection data are available to establish which substrates are preferentially transported by ABCB5. In addition, the length of the protein detected so far (isoform 2) raises unsolved questions about the three-dimensional structure of ABCB5 and its functionality as a transporter.
The implication of other ABC transporters in the melanoma resistance to chemotherapeutic drugs was extensively studied [24]–[28] and remains controversial. We wondered if various transporters might be associated with the selective survival of ABCB5+ cells. This hypothesis was assayed assuming that, if other ABC transporters were co-expressed by ABCB5+ cells, we should expect their concomitant enrichment with ABCB5 after drug treatment. Our data show here that the survival of ABCB5+ cells upon dacarbazine is not associated with ABCB1, ABCC1 or ABCG2 expression. An implication of ABCB1 in the response to drugs known as ABCB1 substrates can not be completely excluded, but does not appear predominant in the survival of ABCB5+ cells. A recent report suggests that a new member of the ABC family, ABCB8, is associated with doxorubicin resistance [29]. A similar expression level of such a transporter in ABCB5+ and ABCB5− cells might explain the lack of differential sensitivity of these cells to doxorubicin but this was not investigated in our study.
Different studies suggest that, in addition to a plasma membrane insertion, some ABC transporters may also be located on subcellular compartments where they play a role in trapping drugs in the cytoplasm before export [30], [31]. The preferential ABCB5 expression in melanocytes and melanomas [11] pointed to a potential link between ABCB5 and the melanosomal compartment in which melanin is synthetized and stored. A role of melanosomes was proposed for melanoma cells chemoresistance to cisplatin [8], [32]. Nevertheless, the WM-266-4 cells lack a functional melanosomal compartment [33], suggesting that the survival of ABCB5+ during dacarbazine treatment is not associated with melanosomes. Moreover, ABCB5+ cells were also found in amelanic tissues [15], [16]. Thus the potential link of ABCB5 with this cellular detoxification pathway remains unclear.
Taken together, our results suggest that anti-melanoma chemotherapy participates to the chemoresistance acquisition that leads to clinical relapse, by selecting tumor cell subpopulations such as ABCB5-expressing cells. This is of particular importance in the chemoresistance occuring upon dacarbazine treatment, but is reinforced in the context of the clinical use of vemurafenib whose strong therapeutic interest is strongly hampered by rapid relapses observed after a few months [7].
ABCB5 appears as a dual potential therapeutic target [34]. On the one hand, the functions of the protein per se may confer tumor cells a survival advantage through the tumor progression and potentially the chemotherapeutic treatment. This last point was recently addressed in colorectal cancer in which ABCB5 was shown to identify a therapy-refractory tumor cell population [16]. On the other hand, the protein ABCB5 might not mechanistically participate to this selective survival, but represents a marker for a subcellular population endowed with intrinsic stem cell-like properties [18]. While the stemness of these cells remains controversial [35], their peculiar properties in terms of tumorigenicity [18], resistance and more recently, immunosuppression [36] remain to be further explored and monitoring the ABCB5+ cells might be an important parameter to evaluate the long term efficiency of melanoma treatment.
The WM-266-4, WM-115, G-361, A-375 and SK-MEL-28 cell lines were obtained from the American Type Culture Collection and the KB and KB-V1 cell lines from the Deutsche Sammlung von Mikrooganismen und Zellkulturen. The WM-266-4, WM-115, G-361, A-375 and KB cells were grown in DMEM (Invitrogen, Cergy Pontoise, France) supplemented with 10% fetal bovine serum (Sigma, Lyon, France), 2 mM glutamine, 100 UI/mL penicillin-streptomycin and 1.25 µg/mL fungizone (both from Invitrogen) in 5% CO2 atmosphere. The SK-MEL-28 cells were grown in MEM (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin-streptomycin, 1.25 µg/mL fungizone, 1 mM sodium pyruvate and 0.1 mM non essential amino acids (all from Invitrogen) in 5% CO2 atmosphere. The KB-V1 cells were grown in MEM (Invitrogen) supplemented with 15% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin-streptomycin, 1.25 µg/mL fungizone, 1 mM sodium pyruvate and 0.1 mM non essential amino acids (all from Invitrogen) and 200 ng/mL vinblastine in 5% CO2 atmosphere. Numerations of viable cells were performed using an Automated Cell Viability Analyzer (Beckman Coulter Vi-Cell).
Flow Cytometry Analysis
The surface expression of ABCB5 and ABCB1 was analyzed respectively with a rabbit polyclonal anti-ABCB5 antibody ABCB5-AbRock (Rockland, Gilbertsville, PA, USA) or a mouse monoclonal antibody ABCB5-AbAbD (clone 3C2-1D12, AbD Serotech, Oxford, UK) and a monoclonal anti-ABCB1 antibody (clone MM4.17, Millipore, Molsheim, France). Cells were detached with 2 mM EDTA in PBS and incubated for 45 min at 4°C with 6 µg/mL of ABCB5-AbRock antibody, 10 µg/mL of ABCB5-AbAbD antibody, 5 µg/mL anti-ABCB1 or corresponding isotype antibodies (Invitrogen). The cells were washed, counter-stained with Alexa-488-conjugated goat anti-rabbit or Alexa-647-conjugated goat anti-mouse Ig antibodies (Invitrogen) and finally incubated with 0.5 µg/mL DAPI (Sigma). The cells were analyzed with a LSRII flow cytometer using the Diva software (both from BD Biosciences, Le Pont-De-Claix, France). ABCB5 and ABCB1 expression were monitored on live cells (gated as DAPI-negative cells).
Si RNA-mediated Down-regulation of ABCB5 Expression
ABCB5 expression was silenced by transfection of siRNA with lipofectamine 2000 Reagent (Invitrogen) after 24 h of serum deprivation. ABCB5 siRNA and the control luciferase siRNA were from Applied Biosystem. The targeted ABCB5 sequence was 5′-ggucggacuacaaucgugg-3′. Cells were analyzed for ABCB5 expression 72 h after the transfection.
RNA was prepared using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) and quantified by the NanoDrop technology (ThermoScientific). Total RNA (1 µg) was reverse transcribed into cDNA with the iScript cDNA Synthesis Kit (Bio-Rad). PCR were performed on an iCycler (Bio-Rad, Marne-la-Coquette, France) using the iQ SYBR Green Supermix (Bio-Rad) kit. All PCR included amplification of HMBS (hydroxymethylbilane synthase) and RPLP0 (ribosomal protein, large, P0) as normalizing controls. The primer sequences were designed as follows: ABCB5 Forward 5′-ccaaatcgggggctgcgcatctgtt-3′ and Reverse 5′-agccgctgctccccacaaatgcta-3′ for ABCB5 isoform 1 and 2; ABCB1 Forward 5′- tgacatttattcaaagttaaaagca-3′ and Reverse 5′- tagacactttatgcaaacatttcaa-3′; ABCC1 Forward 5′- agtggaacccctctctgtttaag-3′ and Reverse 5′- aacagcagcacggtgtagaa-3′; ABCG2 Forward 5′- ccgcgacagtttccaatgacct-3′ and Reverse 5′- gccgaagagctgctgagaactgta-3′; HMBS Forward 5′-atacagacggacagtgtggtgg-3′ and Reverse 5′-ccctgtggtggacatagcaatga-3′; RPLPO Forward 5′-ggcgacctggaagtccaact-3′ and Reverse 5′-ccatcagcaccacagccttc-3′. For each gene, the relative expression ratio was calculated by the ΔΔCt method.
In vivo Study
Animals were handled and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and the European Directive EEC/86/609, under the supervision of authorized investigators. All experiments performed were controlled and approved by the institutional Animal Ethical Committee and by the "Comité Régional d'Ethique pour l'Expérimentation Animale" (Midi Pyrénées, France) (protocol approval number MP/05/16/04/06).
WM-266-4 cells (5×106) were implanted subcutaneously into homozygous female athymic nude mice (Ico:Swiss-nu/nu, Charles River, Saint-Germain-sur-L'Arbresle, France) and allowed to increase to a median value of 100–200 mm3. After randomisation in treatment cages, temozolomide (Molekula, Dorset, UK) was administered i.p. as 4 intermittent injections over 8 days according to a [q2d×3] schedule at 80 mg/kg. In each chemotherapy trial, mice were checked daily, with any adverse clinical reactions noted and deaths recorded. Mice were weighed three times a week during the treatments. Tumors were measured by callipers three times weekly and tumor volumes (mm3) were estimated as = 0.5 (length×width×thickness). Results are presented for experiments involving 3–6 mice per experimental group. T/T0 (%) = mean of (tumor volume on day X of treatment/tumor volume on day 0 of treatment)×100.
The tumors were harvested and dissociated with collagenase (1000 UI/mL, Sigma), elastase (30 UI/mL, Sigma) and DNAse (1 mg/mL, Sigma) in culture medium. After filtration on 70 µm sieves, the cellular suspensions were labelled for cell surface ABCB5 then CD44 using an anti-human CD44 antibody conjugated to Alexa-647 (clone C26, Beckton Dickinson Pharmingen, San Diego, CA, USA) in order to distinguish tumor cells from the infiltrating host cells. ABCB5 labelling was analyzed on live cells (gated as DAPI-negative cells) expressing CD44. In addition, tumor samples from day 17 were paraffin-embedded for immunohistochemistry analyses.
Clinical Cancer Specimens
A total of 15, formalin-fixed paraffin-embedded (FFPE) melanoma tumor samples from 8 patients were obtained after informed consent from the tumor bank of the Department of Pathology, Toulouse-Purpan University Hospital (France). Pathological specimens were collected between 2009 and 2011 and consisted of skin metastases. Tumor samples were collected before (n = 7) and after (n = 8) treatment of the melanoma with monochemotherapy (dacarbazine or fotemustine). All patients presented melanoma metastases at the time the tumor sample was taken (AJCC stage IV). In 3 patients, molecular analysis of the tumor revealed the presence of a BRAF V600E mutation, the mutation was absent in 2 patients. Due to technical artifact, the mutation was not evaluated in 3 patients. In 3 patients, a stable disease was observed after 1 cycle of monochemotherapy (dacarbazine).
For immunohistochemistry, 4-µm sections were deparaffinized and rehydrated. Endogenous peroxide activity was inhibited with a 15 min incubation in 3% (v/v) hydrogen peroxide. An automated staining module (Dako, Glostrup, Denmark) was then used to conduct histological staining. Slides were incubated with a rabbit polyclonal anti-ABCB5 antibody ABCB5-AbSigma (Sigma) followed by a secondary Horse Radish Peroxidase-rabbit antibody and visualized using a colorimetric method (Envision kit, Dako, Glostrup, Denmark). All of the colorimetric immunohistostained sections were analyzed using a Nikon (Eclipse E800) microscope. Semiquantitative double-blind evaluation was conducted by scoring the estimated staining intensity of the ABCB5-labelling.
Cell Treatments with Chemotherapeutic Drugs
The WM-266-4 cells were seeded at 105 cells per well in 24-well culture plates and treated after a 24 h period to allow cell attachment. The cells were harvested for analysis 72 h after the treatment onset and were counted using an Automated Cell Viability Analyzer (Beckman Coulter Vi-Cell) before flow cytometry experiments.
The half maximal effective concentration (EC50) of each drug was determined using a cytotoxicity assay (ATP-lite assay, Perkin Elmer, Courtaboeuf, France). The EC50 on WM-266-4 cells are: 3×10−8 M for vinblastine (Sigma), 3×10−9 M for vindesine (Lilly, Suresnes, France), 3×10−9 M for vincristine (Sigma), 3×10−8 M for paclitaxel (Sigma), 1×10−5 M for dacarbazine (Sigma), 5×10−8 M for doxorubicin (Sigma), 1×10−6 M for etoposide (Sicor, Irvin, CA, USA), 2×10−8 M for gemcitabine (Lilly), 2×10−8 M for camptothecin (Acros, Thermo-Fisher, Illkirch, France) and 4×10−7 M for vemurafenib (Euromedex, Selleckchem).
When mentioned, cycloheximide (10 µg/mL, Sigma) or brefeldin A (10 µg/mL, Sigma) were added respectively 24 h and 4 h before the end-points of the cytotoxic treatments.
Cells were lysed with a Dounce homogenizer at a concentration of 107 cells/mL in the lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 5 mM EGTA, 5 mM NaF, 1 mM NaVO4, 0.1% Nonidet-P40 and 0.1% Triton X114 complemented ex-temporaneously with 1 mM DTT and protease inhibitors cocktail (Calbiochem, Merck Chemical, Nottingham, UK). The unbroken cells and nuclei were pelleted by centrifugation at 600 g for 1 min and washed once with 100 µL of lysis buffer. The post-nuclear supernatant was centrifuged at 72000 g for 30 min. The membrane pellet was solubilised in 2% SDS, 15% glycerol, 50 mM dithiothreitol in 10 mM Tris pH 8, heated at 37°C for 1 h and sonicated. Samples were run on 8% SDS-PAGE and transferred onto polyvinylidene difluoruride membranes. After saturation with 5% dry milk in TBS-0.1%Tween 20, membranes were incubated with either anti-ABCB5 antibody (1/2000 diluted in TBS-0.1%Tween 20-1% BSA) or anti-actin antibody (clone C4, Millipore, 1/1000 in 5% dry milk). After wash, the membranes were revealed with peroxydase-linked secondary goat anti-mouse or rabbit antibody (Jackson ImmunoResearch, Suffolk, UK) and the signals were detected by ECL (GE Healthcare). Band quantification was performed with the Quantity One 1-D Analysis Software (Bio-Rad Laboratories, Inc.) and normalized using actin band intensities.
Data reported are expressed as means ± s.d. of at least three independent experiments. Statistical significance was measured using the Mann and Whitney sum rank test from SigmaStat3.1 software, where p<0.05 was considered to be statistically significant (marked with *) and p<0.01 and p<0.001 was considered highly statistically significant (marked respectively with ** and ***).
Enrichment in ABCB5-expressing cells is associated with protein neo-synthesis and ABCB5 relocation at the cell-surface. WM-266-4 cells were treated with doxorubicin or dacarbazine for 72 h. Cells were either untreated (A) or treated with brefeldin A (B) 4 h before the treatment end-point. A similar experiment was performed except that cells were either untreated (C) or treated with cycloheximide (D) 24 h before the treatment end-point. Cells were labelled for ABCB5 and analyzed by flow cytometry. Histograms show the fluorescence intensity of the ABCB5 cells in representative experiments. The corresponding mean of fluorescence intensity is indicated in each histogram.
Quantification of ABCB5, ABCB1, ABCC1 and ABCG2 mRNA expression after cytotoxic treatments. WM-266-4 cells were treated with various drugs at their EC50 for 72 h. The relative mRNA expression of ABCB5, ABCB1, ABCC1, ABCG2 and HMBS as the house-keeping gene was measured by Q-PCR and the Ct values are reported.
We thank Dr. Chantal Etievant for critical comments and Jean Christophe Blanchet for excellent technical assistance.
Conceived and designed the experiments: MC JR IV BG JPA. Performed the experiments: MC JR AS BG. Analyzed the data: MC JR LL JEG NG JPA. Contributed reagents/materials/analysis tools: LL NM. Wrote the paper: MC JR JPA.
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4. Jilaveanu LB, Aziz SA, Kluger HM (2009) Chemotherapy and biologic therapies for melanoma: do they work? Clin Dermatol 27: 614–625.
5. Garbe C, Peris K, Hauschild A, Saiag P, Middleton M, et al. (2010) Diagnosis and treatment of melanoma: European consensus-based interdisciplinary guideline. Eur J Cancer 46: 270–283.
6. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, et al. (2011) Improved Survival with Vemurafenib in Melanoma with BRAF V600E Mutation. N Eng J of Med 364: 2507–2516.
7. Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, et al. (2011) Dissecting Therapeutic Resistance to RAF Inhibition in Melanoma by Tumor Genomic Profiling. J Clin Oncol 29: 3085–3096.
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The 2017 Pride in your Community 3 Hour Challenge, sponsored by Tim Hortons, will take place on Saturday, June 10. The clean-up will take place from 9 am to 12 noon with a community celebration to follow at the Conception Bay South Arena from 12:30 – 2:00 pm.
Celebration will include Tim Hortons refreshments, hot dogs, a photo booth, live music and family activities. Bags, gloves and a limited number of t-shirts will be provided.
NLEN Member – The Kelligrews Ecological Enhancement Program are participating as a group focusing on the Kelligrews Pond and beach. They meet in the ourdoor classroom on Pond Road.
In 2011, as part of the across-the-board cuts to civil society organizations by the previous federal government, all federal funding to the RCEN and its provincial affiliate networks was cut. This left the national network and most of the affiliates with functioning primarily on a voluntary basis with limited capacity to do their work.
There is hope that the current government will provide for renewed funding in its upcoming budget. This funding is crucial for the survival of the national network and many of the provincial affiliate networks. A proposal has been submitted. It now needs strong and immediate support from environmental groups and individuals across the country.
Please take a few minutes to write to Prime Minister Trudeau and your MP telling them why you value the RCEN, your provincial affiliate network, or environmental networking at the national level in general. Feel free to use the template letter provided below. You can personalize it based on your experience or simply copy and paste.
Let's show our federal politicians that a strong, well-connected grassroots environmental community is essential to a strong Canada!
I am writing to ask that annual core funding to the Canadian Environmental Network (RCEN) be reinstated.
Historically, the RCEN provided a crucial link between environmental groups across the country, both large and small. This link was vital in helping communities address environmental issues right across the country and ensuring a robust approach to the development of environmental policy in Canada.
Since funding was cut in 2011, the RCEN and most of its provincial affiliate networks have been functioning primarily on a voluntary basis with limited capacity to do their work. This is not acceptable. A strong, well-connected grassroots environmental community is essential to a strong Canada. I urge you to reinstate core funding for this crucial work immediately.


text:
Teamwork Makes the Dreamwork
Dr. Marguritte S. Johnson,
Founder & Executive Director
Dr. Marguritte S. Johnson, age 43, resides in Dallas, TX and serves the community as a Social Activist, Founder and Executive Director of two 501(c)(3) nonprofit organizations, Stand 4 Sisterhood and ProActivism Inc.
Johnson recently launched 4CORE: a Multi-generational Mentoring Network committed to the personal and professional development of girls and women.
She is CEO of MSJ-PRO Services, LLC - a consulting agency specializing in formations, filings, and funding strategies for both nonprofit and for profit entities.
Johnson officiated as Executive Pastor for ten years at LIFE Intl' Ministries, and since 2018 has served the advisory board for DISD Innovation Design Entrepreneurship Academy and currently for Bryan Adams High School.
In 2020, she received an Honorary Doctorate in Christian Counseling, in 2021 her Nonprofit Management Certification and in 2022, her Ordination and Licensing in Chaplaincy.
Johnson influences the community as a Speaker, Inspirational Artist, Notary, Best-Selling Author, Panelist, Content Creator, Red Carpet Correspondent, Certified Mental Health First Aid Responder, and Credentialed Mediator.
She celebrates a blissful marriage to Jeremiah Johnson of 24 years and is the proud mother of two, JJ Jr. (age 20) and Mariah Serenity (age 18).
Johnson is passionately intentional about BEING the CHANGE that she desires to SEE in the world TODAY!
Dr. Jeremiah L. Johnson Sr.
Board Treasurer
Dr. Johnson founded a 501(c)(3) non-profit organization called Youth Making A Difference serving low income and at risk youth, empowering them to become productive contributors to society in 1998 and has recently started a new non-profit called Saving Jeremiah, mentoring boys and men in family responsibilities, community engagement, entrepreneurship and Kingdom advancement.
In 2006, he birthed Cookin' For The STARS, a catering entity promoting healthy food preparation utilizing proper cooking utensils; which has now catapulted into his own cookware company, FreshBonAppetit.
Considering his love for marketing and the media industry – Johnson became CEO of Excellence Graphics & Designs in 2011 and in 2012 he established The One Talent Agency to provide local talent with opportunities to be utilized in commercials, movies, print advertisement and documentaries.
Jeremiah currently oversees theHELPstudios, a branding boutique and a multifaceted 6000 sqft. event venue in Farmers Branch, TX.
In 2018, he became an advanced credentialed mediator in the state of Texas via El Centro College and he wholeheartedly accepts his position as Treasurer for both Stand 4 Sisterhood and ProActivism Inc.
Dr. Jeremiah Johnson simply wants the world to know that with God Almighty -All Things Are Possible, and that it is never too late to experience L.I.F.E… Living In Freedom Everyday!
Mariah S. Johnson
Director of Media
Position Needed: Board Secretary
© Stand4Sisterhood. All rights reserved.

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Human Rights Commissioner Kofler on World Water Day
21.03.2019 - Press release
Bärbel Kofler, Federal Government Commissioner for Human Rights Policy and Humanitarian Aid at the Federal Foreign Office, issued the following statement to mark World Water Day on 22 March:
With the 2030 Agenda for Sustainable Development, the international community commited to an ambitious goal in 2015: by 2030, sustainable management and availability of water and sanitation are to be guaranteed for all. Whether women and children in Bangladesh, indigenous peoples in Brazil or refugees in Burundi, we must leave no one behind when it comes to achieving goal 6 on the human right to water and sanitation.
World Water Day 2019 with the motto "Leaving no one behind" is designed to remind us that access to water and sanitation is one of the keys to social, economic and ecological development. Improving water quality and protecting ecosystems are further important subgoals to achieve the sustainability target for water and sanitation. But that is not enough.
We all need to play our part by using water sparingly and attaching due value to it. The Federal Government is doing what it can here in international forums and through its development cooperation. After all, water is our most valuable resource which we need to treat responsibly to ensure all can benefit from it. This is the only way to combat poverty, support sustainable development and ensure that no one gets left behind.
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text:
Google's Pixel Phones: Everything You Need To Know
Michael Nunez
Filed to:October 4
Image: Gizmodo / Google
After months of rumors we're getting our first look at Google's new phones, the Pixel and Pixel XL. The phones are a huge freaking deal for Google, because they are the first flagship Android phones from the company that don't use the "Nexus" brand name. More than a change in name, the phones reflect Google's biggest ideas yet about how you interact with the device you carry everywhere.
The phones still bear many of the same characteristics that you'd find in a classic Nexus device. First, they contain little-to-no wireless carrier modifications (such as a custom UI), making them the most pure version of Android you can buy. They'll also be among the first phones to receive updates when those finally roll out.
Importantly, the phones are the first to use Google Assistant, the company's new artificially intelligent companion that can help Android users search for photos or even make plans. The assistant is similar to Siri, but the crucial difference is that it's powered by Google's search engine, so it can answer more elaborate questions and conduct more complicated tasks.
Google Assistant can be activated on the handsets by saying the words "OK Google," or by simply pressing and holding the home button. Whether you're on your home screen or inside an app, you can ask the assistant for information or help with tasks. Things like "show me my photos from last October" will bring up images stored on your phone. The assistant makes it easier to find specific files, make plans around town, or operate your smart home.
Owners of last year's Nexus 6P and 5X will probably be drawn to the new Pixel phones and immediately notice the fingerprint sensors are once again on the back of the devices. It's a weird design choice that Google introduced on last year's phones, and I'm glad it's sticking around. The rear-facing fingerprint reader somehow feels even more effortless to use than placing your thumb on a home button. It's super easy to get used to.
Both Pixel phones also have a headphone jack, so wired headphone uses can breathe a sigh of relief (at least for now). The phones are basically made to compete directly the iPhone 7 and iPhone 7 Plus, and most of the hardware does. Although the hardware does stack up to the iPhones, the Pixel phones are not waterproof or dustproof, something featured on most flagship Android phones. Both of the new iPhones are also waterproof and dustproof, which makes Google's decision even more peculiar. Although the inclusion of a headphone jack might be enough to sway some people toward the Pixel, but we'd advise you let it go and focus on some of the other attributes.
The new phones get a solid bump in hardware compared to last year's models. The Pixel and Pixel XL both run on a Snapdragon 821 processor, which means they should be able to run 4K video and multiple apps a lot better than the Nexus 6P could with its Snapdragon 810 processor.
The Pixel XL screen is slightly smaller than last year's Nexus 6P (5.5 inches and 5.7 inches respectively), but it manages to display the same resolution (1440 x 2560) and even packs in a higher pixel density, which should mean it emits a sharper picture on a smaller screen.
The smaller Pixel phone only has a 5-inch screen that displays 1920 x 1080 resolution, which is nearly identical to last year's Nexus 5X. The Pixel does have a slightly higher pixel density, but the difference is so marginal, we don't expect it to have any bearing on the look of the screen.
It will be interesting to see how the cameras on these new phones stack up to last year's models in our hands-on tests. Both Pixel phones come with a 12-megapixel rear camera that appears to be good enough for most average users.
The Pixel phone cameras shouldn't be as good in low light as the iPhone or Galaxy S7 because they only have a f/2.0 lens (for comparison, the iPhone 7 has a f/1.8 and the Galaxy S7 has a f/1.7).
The specs haven't prevented Google from touting the camera's abilities. The Pixel phones have apparently earned an 89 DXO Mark Mobile rating, the highest of any mobile phone ever. Google claims it's the best camera phone ever made, but we'll believe it when we see it.
One of the last things you'll notice about the Pixel phones is that they both use USB-C ports for chargers, something that's still quite rare. Although dozens of products already rely on USB-C for charging purposes, two of the most popular smartphones—the Galaxy S7 and iPhone 7—do not. The fact that these new phones use USB-C will definitely help cement the new standard and get more people to start using it.
In a huge bummer, the phones will be exclusive to Verizon, which means they'll probably fall short of the kind of mainstream appeal Google is hoping for. The sliver lining is that Google will offer the Pixel unlocked in the Google Store. The Pixel phones come with a hefty new price tag, which is also a departure for Google. The phones start at $650, and they're available for pre-order today.
Technology editor at Gizmodo.
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NFR Licenses for VMware Professionals - T.B.D.
Update (2016/05/18): I've created an NFR License page, so that it's easier for people to find the list and easier for me to update it. The table below may no longer be accurate, so please visit the page for the latest information.
I've recently come across a few vendor blog posts about free NFR licenses for VMware professionals. Not For Resale (NFR) licenses are a specific license that vendors use to make sure software or products are not (re)sold. They're usually given to people to use the products for testing, evaluation and education.
Vendors recognize value in letting IT professionals have hands on experience with their products. In an educational setting, such as a test lab or home lab, these pros can learn the ins and outs of a solution. Sometimes IT pros don't have access to products as they're not available from work or too expensive. NFR licensing can help overcome these limitations.
Here I will list all the NFR licenses I've found that are available to VMware professionals. A vendor usually recognizes someone as a VMware professional if they are VMware certified or are a VMware vExpert. Membership or leadership in your local VMUG is sometimes enough as well.
The product name will link to the product page or blog post (and in some cases social media post!) that references the vendor's NFR offer, including license and conditions. Make sure you read the vendor's EULA for specific product usage, conditions and restrictions.
*Link is only available to current vExperts.
If you have any updates let me know in the comments or on Twitter.
Edit: I've added Veeam ONE to the list. Turns out when you request your NFR license for Veeam Backup & Restore you also get a Veeam ONE NFR license.
Edit: Thanks to Chris Colotti and Jeramiah Dooley, vExperts can try out vCHS free for 30 days.
Revello recently announced free Ravello Lab Service for vExperts. 1,000 hours of service per month, use-it-or-lose-it, for one year. See the link in the post for details.
No sooner had I gotten an update out the door than another item shows up to add to the list!
RT @deeabson: ICYMI: Not For Resale (NFR) Licenses for VMware Professionals http://t.co/HHGPmMHINt < Major update!
Thycotic – New offer for 1 year, 10 user NFR license of Secret Server Professional Edition.
Royal Applications – Updated offer for an NFR license of Royal TS/X for personal use (new URL & company name).
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Recent advancements in material science exploit non-covalent interactions, such as halogen bonding (XB) or π-stacking within solid-state molecular frameworks for application in organic electronic devices. Herein, we focus on these and other non-covalent interactions and the effect that furan and thiophene substituents play on the solid-state properties of co-crystals formed between pentafluoro(iodoethynyl)benzene (F5BAI; XB donor) and a pyridine disubstituted with either furans or thiophenes (PyrFur2 and PyrThio2; XB acceptors). Spectroscopic and thermal analyses of 1 : 1 mixtures provide indirect evidence of XB interactions, whereas X-ray crystallography provides direct evidence that XB and π-stacking are present in both co-crystals. Density functional theory (DFT) computations provide insight into the relative electronic energetics of each pair-wise contact observed in the experimental F5BAI-PyrFur2 and F5BAI-PyrThio2 co-crystals.


text:
Q: Click on Anchor tag automatically via C# Code(Windows desktop application) Click

I have this link in my page , i need to click automatically when i traverse the doc,
After click it show some information the Div, I want to Fetch that information..
So i need this.. Anyone can help me ...

A: using (var browser = new IE("Your URI)")
{
    browser.Link(linkId).Click();
    var div = browser.Div(divId);
}

redpajama_set_name: RedPajamaStackExchange


Payaso Cheese Puffs are a delicious way to entertain your guests in a party, use at the beach, the park, during work, lunchtime. They are fresh and crunchy with the added zest of flavor. This product is baked ,not fried. Enjoy.


text:
Honey production in Israel — a sweet undertaking
BY BATSHEVA POMERANTZ, IPS
Honey is a popular ingredient in Rosh Hashana dishes, and the greeting Shana tova u'metuka ("A good and sweet year") is on everyone's lips. At the festive Rosh Hashana meal, a piece of challah trickled with honey is followed by a slice of apple dipped in honey, and honey cake tops off the feast as a classic dessert.
Due to increased local demand for honey in recent years, the proverbial Land of Milk and Honey (whose 90,000 beehives produce a whopping 3,600 tons of honey annually) has found itself in the unlikely situation of having to import honey to maintain its honey industry.
To solve the problem, the Jewish National Fund earmarked forests for beehives and agreed to provide the Honey Council with some 100,000 nectar-producing saplings and plants each year. When this didn't suffice, the Jewish National Fund, together with the country's 500 apiarists, took further action and in early 2006 implemented the planting of some 200,000 additional trees as a source of nectar for bees. Beekeepers can now travel with their beehives to one of 6,300 pasture points situated around the country.
The raising of honey bees first took off in the Land of Israel in the late 19th century, although in the biblical era dates were made into honey, and many believe the notion of a "land flowing with milk and honey" actually referred to date honey.
Fifth-generation apiarist Malka Ben Zeev of the Dvorat HaTavor Land of Silk and Honey Center in the Lower Galilee can trace her profession back to her Hungarian ancestors. Her agronomist husband, Yigal, learned the art of rearing silkworms in Iran in the late 1960s while an emissary of the Israeli government. The two joined forces, and in 1970, the newlyweds established the only silkworm breeding farm in Israel and began developing a successful honey production business at Moshav Shadmot Dvora, a stone's throw from Mount Tabor and the second century BCE Silk Road.
Dvorat HaTavor, which is recognized by the Israel government as an agro-tourism attraction, enjoys visits from students of all ages, retirees and groups of young people from the Diaspora. A program in Arabic is geared to the Arab residents of the Galilee.
The Visitors Center employs Galilee residents — often recently discharged IDF soldiers — as guides to explain about removal of the combs and extraction of the honey from the hives, each of which produces approximately 45 kilograms of honey a year.
"We place our hives in Galilee fields where there are natural wild flowers," Malka explains, "and avoid orange or avocado orchards that are sprayed with pesticides. The bees graze on white forest flowers according to the season. In the summer our honey is produced from thistles."
Israel has two to three blossom seasons when the bees suck the nectar from more than one hundred types of plants, starting off the fascinating production process. The plants are divided into cultivated plants, like citrus (from which most of the honey is produced in Israel), and wild flowers. The flowers from which the nectar is collected affect the flavor and color of the honey. Plants include avocado, marjoram, eucalyptus, thorns, citrus, three-lobed sage, thyme and clover.
Some types of honey, like those made from avocado and eucalyptus nectar, are suitable for roasting meat, while others, from the nectar of wild flowers, are suited for light repasts consisting of leafy salads, fruit and yogurt. Honey produced from citrus is suitable for baking cakes and dishes with a dough base.
Honey was used for thousands of years for health purposes and its long shelf life has been proven by the discovery of jars of still edible honey entombed with Egyptian mummies. According to the Honey Council, honey is a sterile natural food and can be effective in warding off winter illnesses, boosting appetite and energy. As a sweetener, honey is 25 percent less caloric than sugar, with 25-30 calories per teaspoonful.
Healthy byproducts of the beehive include propolis, "the natural antibiotic," which protects the beehive from illness. It is scraped from each hive in small quantities and used in anti-fungus creams, creams to soothe the skin and sprays for irritated throats. Another healthful byproduct is pollen, which is used to nourish the bees, and which is rich in vitamins, including vitamin B-12. "Propolis and pollen are expensive because of the intensive work needed to extract them from the beehive," notes Malka.
Finally, let us not forget the hardworking bees, who must visit more than 4.5 million flowers and travel over 140,000 kilometers to produce a single kilogram of honey so that we may enjoy this traditional treat on Rosh Hashana.
Note: In 2004, the Apiarists Organization in Israel changed the name of its profession. Until then an apiarist was known as a kavran, which, although spelled differently, sounded identical to the word for undertaker in Hebrew. An Israeli apiarist is now called a dvorai, from dvora (bee).
Do Paul Rudd and Alicia Silverstone's Super Bowl ads hold the key to eternal youth?
This online kosher nuts business actually started as a pushcart
From Mel Brooks to Michael Douglas: Producer Julian Schlossberg writes memoir about working with Jewish stars over 6 decades
My "rock 'n roll rabbi is following Springsteen during a mini-sabbatical
Bruce Springsteen has a special connection with his Jewish fans


Learn about Diana Matheson's work with Right To Play, and the Play Program's work with aboriginal communities in Canada.
Diana works with Right To Play's Play Program, working in Aboriginal communities in Canada.
The PLAY program works with its partner communities and organizations to create meaningful and lasting change in four key areas, so that children and youth are experiencing better education outcomes, better health, healthier relationships, and higher youth employment.


Back to school after Spring Break and sleeping in for a week (well, some of us slept in) — I think this song might be needed to get boys moving this morning!
From our late friend's cereal bowl.
This entry was posted in music, Musical Monday, That's How We Roll. Bookmark the permalink.
So sweet – but I'm not looking forward to school and work… The Easter hols have been delightful!
Great basket of flowers. Is it time to plant there? Cute song – I didn't listen, just read the lyrics (headache).
I am barely upright after a mere 5 hours sleep. Is it possible I am not young anymore.
I have never heard this song before and I am laughing at the silliness and totally impressed at the same time. Thanks for sharing — I think.


text:
Q: Как использовать button с переменной? Я не хочу писать большого кода,кнопки уже созданы. Как заменить?
button4->Visible=false;
button5->Visible=false;
button6->Visible=false;
button7->Visible=false;
button8->Visible=false;

на
for (i = 4; i < 9; i++) {
    button i->Visible = false;
}

Помогите пожалуйста.
КНОПКИ СОЗДАНЫ В RUN-TIME
A: Что-то в последнее время подобные вопросы часто стали тут появляться.
Нет, в С++ так делать нельзя. Надо указатели на динамически созданные объекты (в данном случае - объекты кнопок) группировать в массивы или (намного лучше) в контейнеры, например, vector, и далее обращаться к ним через индекс.


For years I dismissed greens. Most people tend to over cook them, making them a soggy, gooey mess.
But in my first season with my share at A Place on Eearth CSA, I had to face the greens. Kale. Big bunches of it every week. I discovered something odd... I love kale, especially raw kale (and, oddly enough, so do my dogs). Yeah, you read that right. Raw kale. And yes, you read the parentehses right, too: my dogs LOVE kale. They'll eat anything.
My husband is not quite the kale devotee that I (or the dogs) became, even when lightly steamed and wilted with some garlic and salt (yum!). But, thanks to the indispensible Simply In Season cookbook, I've figured out how to get him to eat the kale.
Brown the sausage thoroughly, drain most of the fat that cooks off of it, but reserve about 2 TBSP.
Heat the reserved fat in a big soup pot (about 4-5 quarts), add the onions and garlic (or garlic scapes), and sautee until soft (about 5 minutes). If your sausage didn't throw off much fat, just toss in some olive oil, enough to keep the onions from sticking.
Add the sausage, potatoes and stock. Heat to boiling. Reduce heat and simmer for about 15 minutes, or until potatoes are tender.
Add the greens and condensed milk, and cook a few minutes longer. If using spinach, probably about one or two minutes will suffice. I recommend about 5 minutes for the kale.
Serve with some nice crusty dunkin' bread.
The original recipe is, of course, much more precise (y'all know I don't really LIKE measuring things, right?), and calls for salt and pepper. If you use the Italian sausage you won't need any additional spice. The spice in the sausage will be plenty.
Again, I highly, HIGHLY recommend Simply in Season. It's a fantastic cookbook, and should be a mainstay in any collection. Great for learning to eat seasonally, and the recipes are incredibly well organized.


Majestic castles rise under your hands, the fairy-tale heroes smile, and magnificent landscapes flaunted! But not only children like sculpting fanciful sculptures. Often it is created by adults to get into the Guinness Book of Records.
The Sand Sculpting event used to be held in Frankston, one of the districts of Melbourne. From 2018, it moved to the Boneo Maze, about an hour drive from Melbourne. Sand sculptures take over the area at the height of the Australian summer (December-April). Sculptors from all over the world are turning tonnes of sand into masterpieces. The size of the sand figures is very different, from the size of a soccer ball to truly gigantic figures.
The theme of the Sand Sculpting Australia changes from year to year: toys, fairy tales, dinosaurs, famous personalities. For children, organisers provide master classes where they can learn how to cut out sand. Adults and kids form sand castles and participate in fun tournaments and interactive games held during the festival.


text:
Q: ConvertAPI/PHP code upload/download 0 bytes I'm trying to call the ConvertAPI RESTful API using PHP/CURL to convert a form upload PDF to PNG. The file upload is working as the file is showing up on the server, but nothing is returned with curl_getinfo showing [size_upload] => 0 and [size_download] => 0. Can someone point out the error in my code?
define("UPLOAD_DIR", "./tmp/");
$rest_url="https://do.convertapi.com/Pdf2Image";

$up_file=UPLOAD_DIR.rand(100,999)."_".basename($_FILES['fileField']['name']);
if (!move_uploaded_file($_FILES['fileField']['tmp_name'], $up_file)) {
    die("Possible file upload attack!");
}

$cfile=new CURLFile($up_file);

$params=array(
    "ApiKey" => 11111111,
    "OutputFormat" => "png",
    "OutputFileName" => "converted.png",
    "File" => $cfile
    );

$ch=curl_init($rest_url);
curl_setopt($ch, CURLOPT_POST, 1);
curl_setopt($ch, CURLOPT_HEADER, 1);
curl_setopt($ch, CURLOPT_RETURNTRANSFER, 1);
curl_setopt($ch, CURLOPT_POSTFIELDS, $params);
$res=curl_exec($ch);
$headers=curl_getinfo($ch);
curl_close($ch);

echo "<pre>";
print_r($headers);
var_dump($res);

A: The upload stops if the URL is written with https://. It should be http:// instead (without the SSL).
Use this:
    $rest_url="http://do.convertapi.com/Pdf2Image";

Instead of:
    $rest_url="https://do.convertapi.com/Pdf2Image";


text:
Statement on cuts to Employment & Support Allowance (ESA)
I am committed to fighting this Government's draconian cuts upon the most vulnerable. I have consistently voted against the cuts however unfortunately I've missed one vote due to illness but I've challenged these cuts time and again.
Tenth Delegation Legislation Committee: Draft Tax Credits (Income Thresholds and Determination of Rates) (Amendment) Regulations
I sat on the Delegated Legislation committee concerning Government cuts to tax credits. Just last week I challenged the Government over equality impact assessments and in particular the disproportionate impact that changes to tax credits will have on women and children.
Dawn Butler (Brent Central) (Lab):
Is my hon. Friend aware of the extent of the impact assessment that the Government have undertaken on these new limits?
As there is no impact assessment, does the Minister agree that the measure will probably affect women and children more than any other group?
Available in full online at http://www.publications.parliament.uk/pa/cm201516/cmgeneral/deleg10/160303/160303s01.htm
Treasury Questions: Employment & Support Allowance
One of the very first things I did after being returned to Parliament last year was to raise the issue of the proposed cuts to Employment & Support Allowance (ESA) directly with the Chief Secretary to the Treasury, Greg Hands MP on 21st July 2015. See below an excerpt of the Hansard:
What discussions he has had with the Secretary of State for Work and Pensions on the effect of proposed changes to employment support allowance on levels of employment. [901118]
The Chief Secretary to the Treasury (Greg Hands):
We created 2 million jobs in the previous Parliament, and our objective is to create a further 2 million in this Parliament. A crucial part of that is the welfare reforms that we have introduced to help make work pay, which is consistent with our long-term economic plan.
Dawn Butler:
Of course we all want to see work pay, but a large section of the community are sometimes unable to work for short periods of time because of illnesses such as sickle cell disease. The Minister seems to have overlooked that group of people.
Greg Hands:
I thank the hon. Lady for her question. Our welfare reforms are based on the principle of fairness; fair on those who receive the benefits and fair on those who pay the tax. With regard to a specific group, there is clearly a difference between the work-related activity group and the support group, and we are happy to look at those differences. She is clearly not satisfied with what we are doing, but she is also one of the 48 Labour Members who rebelled last night on welfare, so I do not think that she is satisfied with her Front Benchers' position either.
Available in full online at http://www.publications.parliament.uk/pa/cm201516/cmhansrd/cm150721/debtext/150721-0001.htm#15072125001820.
Welfare Reform & Work Bill
When the Welfare Reform & Work Bill came before the House of Commons for its third reading I was one of only 48 MP's to vote against the bill which included the Government's cuts to ESA. In that debate I highlighted the disproportionate impact of the Government's cuts on children in Brent Central. See below an excerpt of the Hansard:
On the effect of the cuts, Brent council has produced its own report, which highlights the fact that in Brent 13,600 households and 26,200 children will be affected.
Dr Whiteford (SNP):
The hon. Lady makes a useful point. I am aware that Brent is one of the areas where the benefits cap will be particularly keenly felt, but all our big conurbations are affected, especially those where there is a large gap between the incomes of the wealthiest and people who are earning what in any other part of the country would be a decent wage, but in certain parts of the UK is not enough to live on.
I am glad to see that Labour Members have supported amendment 56, which I intend to press to a vote this evening. I shall also address some of the related amendments, 57 to 65, all of which would affect support for those distanced from the labour market, whether under employment and support allowance or universal credit. They would remove the provisions in the Bill that seek to reduce ESA for those in receipt of the work-related activity component.
I want to be absolutely clear that SNP MPs will oppose the proposals in clauses 13 and 14, which are an outright attack on people who are seriously sick, disabled, or living with debilitating long-term health problems. We are talking about people who are so seriously incapacitated that even the Government's own stringent assessment process has deemed them unfit for work at present. Slashing support for sick people will not help them recover more quickly. In fact, money worries are one of the things that often slow down people's recovery from serious illness. We have just heard a powerful speech delivered from the Government Benches about support for people who are terminally ill, but sometimes people recovering from illnesses that could go either way need a long time to recover, and they do not always get the support and the sympathy they need.


text:
Q: Get location name from fetched coordinates get the name I have found this solution: here to get the location name but I don't know how to display it in my textView:
    LocationManager locationManager = (LocationManager)getSystemService(Context.LOCATION_SERVICE);
String provider = locationManager.getBestProvider(new Criteria(), true);

Location locations = locationManager.getLastKnownLocation(provider);
List<String>  providerList = locationManager.getAllProviders();
if(null!=locations && null!=providerList && providerList.size()>0){
double longitude = locations.getLongitude();
double latitude = locations.getLatitude();
Geocoder geocoder = new Geocoder(getApplicationContext(), Locale.getDefault());
try {
    List<Address> listAddresses = geocoder.getFromLocation(latitude, longitude, 1);
    if(null!=listAddresses&&listAddresses.size()>0){
        String _Location = listAddresses.get(0).getAddressLine(0);
    }
} catch (IOException e) {
    e.printStackTrace();
    t.append("\n " + ????? + " ");
}

}

The t is the textView

A: You have the name address in the String _Location. Therefore, you could set the value of TextView in the try block.
try {
List<Address> listAddresses = geocoder.getFromLocation(latitude, longitude, 1);
if(null!=listAddresses&&listAddresses.size()>0){
    String _Location = listAddresses.get(0).getAddressLine(0);
}
t.setText(_Location);
}

Further Infomation
    String address1 = listAddresses.get(0).getAddressLine(0);
    String city = listAddresses.get(0).getLocality();
    String state = listAddresses.get(0).getAdminArea();
    String country = listAddresses.get(0).getCountryName();
    String postalCode = listAddresses.get(0).getPostalCode();
    String knownName = listAddresses.get(0).getFeatureName();

    t.setText(address1 + " " + city + "\n" + state + " " + postalCode);

You could use this code to get further information about the adress


At the time of the last Lintian run, the following possible problems were found in packages maintained by David Watson <dwatson@debian.org>, listed by source package. See also the report showing only errors and warnings. Also see their QA overview.
"An online contract bridge game. Gtk client"