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Home > Vol 5, No 17 >	Morimoto
Annexin A4 induces platinum resistance in a chloride-and calcium-dependent manner
Akiko Morimoto, Satoshi Serada, Takayuki Enomoto, Ayako Kim, Shinya Matsuzaki, Tsuyoshi Takahashi, Yutaka Ueda, Kiyoshi Yoshino, Masami Fujita, Minoru Fujimoto, Tadashi Kimura and Tetsuji Naka _
Akiko Morimoto1,2, Satoshi Serada2, Takayuki Enomoto3, Ayako Kim2, Shinya Matsuzaki1 ,Tsuyoshi Takahashi2,4 ,Yutaka Ueda1, Kiyoshi Yoshino1, Masami Fujita1, Minoru Fujimoto2, Tadashi Kimura1 and Tetsuji Naka2
1 Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Japan
2 Laboratory for Immune Signals, National Institute of Biomedical Innovation, Japan
3 Department of Obstetrics and Gynecology, Niigata University Medical School, Japan
4 Department of Surgery, Osaka University Graduate School of Medicine, Japan
Correspondence:
Tetsuji Naka , email:
Keywords: Annexin A4; platinum resistance; annexin repeat; chloride ion
Received: April 04, 2014 Accepted: August 03, 2014 Published: August 04, 2014
Platinum resistance has long been a major issue in the treatment of various cancers. We previously reported that enhanced annexin A4 (ANXA4) expression, a Ca2+-regulated phospholipid-binding protein, induces chemoresistance to platinum-based drugs. In this study, we investigated the role of annexin repeats, a conserved structure of all the annexin family, responsible for platinum-resistance as well as the effect of knockdown of ANXA4. ANXA4 knockdown increased sensitivity to platinum-based drugs both in vitro and in vivo. To identify the domain responsible for chemoresistance, ANXA4 deletion mutants were constructed by deleting annexin repeats one by one from the C terminus. Platinum resistance was induced both in vitro and in vivo in cells expressing either full-length ANXA4 or the deletion mutants, containing at least one intact annexin repeat. However, cells expressing the mutant without any calcium-binding sites in the annexin repeated sequence, which is essential for ANXA4 translocation from the cytosol to plasma membrane, failed to acquire platinum resistance. After cisplatin treatment, the intracellular chloride ion concentration, whose channel is partly regulated by ANXA4, significantly increased in the platinum-resistant cells. These findings indicate that the calcium-binding site in the annexin repeat induces chemoresistance to the platinum-based drug by elevating the intracellular chloride concentration.
Since cisplatin was first introduced as an anticancer drug in the 1970s [1], various platinum-based drugs have been developed and widely used not only against gynecological but also against other cancers, including lung, colorectal, testicular, prostate and bladder cancer [2-6]. Although these platinum-based drugs have significantly contributed to improve survival rates, chemoresistance to these drugs has become a major problem in recent years [7-9]. It has now been elucidated that the mechanism of platinum resistance is mediated by reduced platinum accumulation, increased platinum detoxification, increased repair of platinum–DNA adducts and inhibited apoptosis [10-12]. Several proteins have been reported to be candidate factors such as copper transporters: CTR1[13], ATP7A and ATP7B[14-17]; multidrug resistance protein 2 (MRP2) [18-20]; glutathione S-transferase enzyme π (GSTπ) [21]; excision cross-complementing gene 1 (ERCC1) [22]; receptor-interacting protein 1 (RIP1) [23]; microRNAs [24-26]; and p53 [27]. In contrast, there are still several proteins related to platinum resistance without a full understanding of how these proteins help cells to confer platinum-based drugs.
We recently reported that annexin A4 (ANXA4) is overexpressed in ovarian clear cell carcinoma and induces chemoresistance to platinum-based drugs [28]. Annexins are calcium-regulated and negatively charged phospholipid membrane-binding proteins. The basic structure of annexins consists of 2 major domains: a conserved structural element called an annexin repeat, a segment of 70 amino acid residues at the C terminus, and the N-terminal region unique for a given member of the family and determining individual annexin properties in vivo. The annexin repeat possesses the calcium and membrane binding sites and is responsible for mediating the canonical membrane binding properties [29]. These domains in ANXA4 are surrounded by relatively short amino and carboxy termini that do not have any known function [30]. ANXA4 is involved in membrane permeability, exocytosis and regulation of chloride channels in a calcium-dependent manner [29, 31-33]. ANXA4 is almost exclusively expressed in epithelial cells [34]. With regard to cancer, ANXA4 overexpression has been reported in various tumours, such as lung, gastric, colorectal, renal, pancreatic, ovarian and prostate cancer [28, 35-39] and is associated with tumour invasiveness, metastasis and chemoresistance [37, 40]. Moreover, ANXA4 has been shown to be associated with resistance to platinum-based drugs [28, 41-43].
ANXA4-induced platinum resistance appears to be mediated in part by the increased extracellular efflux of platinum mediated by the copper transporter ATP7A[28, 44]. Another mechanism of ANXA4-induced chemoresistance is the modulation of NF-κB transcriptional activity [45]. ANXA4 suppresses NF-κB transcriptional activity through interaction with the p50 subunit in a calcium-dependent manner; ANXA4 causes resistance to apoptosis induction by etoposide.
While ANXA4 prominently associated with chemoresistance, the functional domain of ANXA4 remains unclear. Therefore, to clarify the functional domain of ANXA4 is required to understand detailed mechanisms of the chemoresistance induced by ANXA4 and also overcome chemoresistance. In this study, focusing on platinum resistance, we aimed to identify the ANXA4 domain relevant to chemoresistance with regard to its structure as well as to test whether knockdown of ANXA4 expression could improve platinum resistance. Our data showed that the annexin repeat plays an important role in platinum resistance induced by ANXA4, which occurs in a calcium-dependent manner.
Establishment of ANXA4 knockdown RMG-I cells
To create cell lines with a stable ANXA4 knockdown, we analysed ANXA4 expression in ovarian cancer cells using western blotting. ANXA4 expression was strong in clear cell carcinoma cell lines (OVTOKO, OVISE and RMG-I) compared with serous adenocarcinoma cell lines (A2780, OVCAR3 and OVSAHO) and a mucinous adenocarcinoma cell line (MCAS; Fig. 1A). To see whether blocking ANXA4 expression was a valid chemosensitising strategy for ovarian clear cell carcinoma treatment, ANXA4 was stably suppressed using an ANXA4 shRNA plasmid. We established RMG-I-Y4 and R5 cell clones as well as RMG-I NC7 cell clones transfected with the empty vector as a control. Compared with RMG-I NC7 and untransfected control parent RMG-I cells, ANXA4 expression was markedly down-regulated at the protein level in RMG-I-Y4 and RMG-I-R5 cells (Fig. 1B). In the absence of any drug treatment, the growth rate among the 4 cell lines was similar in vitro (data not shown).
Fig.1: Knockdown of ANXA4 expression attenuates platinum resistance. (A) ANXA4 expression in indicated ovarian cancer cell lines and (B) established ANXA4 knockdown RMG-I cells (R5 and Y4) was confirmed using Western blotting. (C) IC50 for both cisplatin and carboplatin was significantly reduced in R5 and Y4 cells compared with NC7 cells. Data are presented as mean ± SD (*p < 0.01).
Knockdown of ANXA4 expression enhances sensitivity to cisplatin and carboplatin
The sensitivity to cisplatin and carboplatin was assessed in the 3 RMG-I clones NC7, R5 and Y4. Compared with the IC50 for cisplatin in NC7 cells, IC50 was significantly decreased in Y4 cells and R5 cells (p < 0.01; Fig. 1C, left panel). Similarly, IC50 for carboplatin significantly decreased in Y4 cells and R5 cells compared with NC7 cells (p < 0.01; Fig. 1C, right panel). IC50 for cisplatin and carboplatin decreased approximately 2-fold because of the knockdown of ANXA4 expression.
Suppression of ANXA4 expression improves platinum sensitivity in vivo
To determine whether ANXA4 knockdown in clear cell carcinoma cells improved platinum sensitivity in vivo, NC7 and Y4 cells were subcutaneously injected in ICR nu/nu mice. One week after inoculation with the tumour cells, the mice were randomised into 2 groups and received cisplatin or PBS i.p. twice a week for 4 weeks. The tumour growth rate in the absence of drugs was similar for both cell lines (Figs. 2A and 2B). Cisplatin treatment had very little effect on NC7 cells (Fig. 2A), but tumour volume markedly decreased in Y4 cells (Fig. 2B). Cisplatin treatment significantly decreased tumour growth in Y4 cells (87.4 ± 1.8%) compared with NC7 cells (-1.1 ± 18.0%; p < 0.01; Fig. 2C). These results showed that ANXA4 knockdown in the RMG-I cell line significantly attenuated resistance to cisplatin in vivo.
Fig.2: ANXA4 knockdown cells show enhanced sensitivity to cisplatin in vivo. Female ICR nu/nu mice were subcutaneously inoculated with RMG-I NC7 or Y4 cells and given PBS (control group: filled circles) or cisplatin i.p. (3 mg/kg; treatment group: open circles) twice weekly for 4 weeks (n = 6 per group). Growth curves of NC7 tumours (A) and Y4 tumours (B). The mean volume (points) ± SE (bars) is shown. (C) Comparison of the cisplatin-induced growth inhibition of tumours 46 days after treatment among NC7 and Y4 tumours. The average (columns) ± SE (bars) are shown (*p < 0.01).
The annexin repeat domain is required for the platinum drug resistance
To identify a possible resistance-related domain within the annexin repeated sequence of ANXA4, we constructed 3 deletion mutants by deleting the annexin repeats one by one from the C-terminal region. Figure 3A shows the structure of each deletion mutant. Full-length ANXA4, 3 ANXA4 deletion mutants or the empty vector were transfected into NUGC3 cells, whose endogenous ANXA4 expression is relatively low (Supplementary Fig. S1). Therefore, we established cell lines stably overexpressing full-length ANXA4 (FL-22), each ANXA4 deletion mutant (R3-6, R2-13 or R1-12) or the empty vector (NC-14). Expression of each ANXA4 deletion mutant was confirmed using Western blotting (Fig. 3B).
Subsequently, the sensitivity to the platinum-based drugs cisplatin and carboplatin was assessed. Cells transfected with full-length ANXA4 and the 3 deletion ANXA4 mutants were significantly more resistant to both cisplatin and carboplatin compared with control cells, approximately with a 1.7- to 2.2-fold increase in IC50 for cisplatin (p < 0.01) and a 1.4- to 1.7-fold increase in IC50 for carboplatin (p < 0.05; Fig. 3C).
To test whether these deletion mutants induce platinum resistance through regulating cellular drug concentration as previously reported [28], we quantitated the intracellular platinum content of each deletion mutant-transfected cell clone after cisplatin treatment, which is one of the most representative platinum drugs. Platinum accumulation was significantly reduced in cells overexpressing either full-length ANXA4 or any of the 3 deletion mutants compared with NC-14 cells regardless of the incubation time after cisplatin exposure (Fig. 3D). These results suggested that the decreased intracellular platinum contents were associated with platinum resistance of the cells transfected with ANXA4 full length and each deletion mutant.
Fig.3: Annexin repeat domain is required for the platinum drug resistance. (A) A structural map of ANXA4 and 3 deletion mutant proteins. Annexin repeats were deleted one by one from the C-terminal site. (B) Established deletion mutant cells together with parent cells, control cells and RMG-I as a positive control were confirmed using Western blotting. (C) Compared with NC-14 cells, IC50 for both cisplatin and carboplatin was significantly increased in FL-22 and all other mutant cells. (D) Intracellular platinum accumulation after treatment with 100 µM cisplatin for 60 min with or without additional 3 hr of incubation in cisplatin-free medium. Data are presented as mean ± SD (*p < 0.05, **p < 0.01).
The calcium-binding site of the annexin repeat is responsible for platinum resistance
As specified above, platinum resistance was enhanced in cells overexpressing ANXA4 deletion mutants, which contained at least 1 intact annexin repeat. Thus, to assess whether the calcium-binding site of the annexin repeat sequence was involved in chemoresistance, another deletion mutant, R1(E70A) was constructed. Within the annexin repeat next to the N-terminal region, the 70th amino acid, glutamic acid, was responsible for the calcium-dependent activity of ANXA4 [30]. Accordingly, at this site, the point mutation variant of R1, R1(E70A), loses the function of its calcium-binding site (Fig. 4A). Similar to other deletion mutants, R1(E70A) was transfected into NUGC3 cells and designated R1(E70A)-95. Western blotting revealed that R1-12 had the same molecular weight as R1(E70A)-95 (Fig. 4B). R1(E70A)-95 did not induce resistance to either cisplatin or carboplatin (Fig. 4C). Moreover, the intracellular platinum content of R1(E70A)-95-transfected cells did not decrease compared with that of NC-14 cells after 0 hr or 3 hr of additional incubation in cisplatin-free medium (Fig. 4D). According to the above results, the platinum resistance of ANXA4 seemed to be related to the calcium-binding site of the annexin repeat next to the N-terminal domain.
Fig.4: The calcium-binding site of the annexin repeat is responsible for induction of the platinum resistance. (A) A structural map of R1 and R1(E70A) variants without a function for the calcium-binding site. (B) Western blotting confirmed the mutant established cell lines. (C) Unlike R1-12, the R1(E70A)-95 cell clone was not resistant to either cisplatin or carboplatin. (D) Intracellular platinum accumulation in R1(E70A) cells was the same as in NC14 cells both with or without additional 3 hr of incubation in cisplatin-free medium. Data are presented as mean ± SD (*p < 0.05, **p < 0.01).
The calcium-binding site of the annexin repeated sequence is required for the resistance to platinum-based drugs in vivo
To determine whether the annexin deletion mutants of ANXA4 influenced the sensitivity to cisplatin in vivo, we inoculated ICR nu/nu mice with NC-14, FL-22, R1-12 or R1(E70A)-95 cells. Mice in each group were randomised into 2 subgroups and received either cisplatin at 3 mg/(kg∙d) or PBS i.p. once a week for 3 weeks. Cisplatin markedly decreased tumour volume in mice injected with NC-14 and R1(E70A)-95 cells, whereas the treatment effect was relatively smaller in mice injected with FL-22 and R1-12 cells (Fig. 5A). Consistent with the tumour volume, tumour growth was significantly inhibited by cisplatin in mice inoculated with NC-14 (96.5 ± 1.3%) and R1(E70A)-95 cells (87.8 ± 11.4%) compared with those injected with FL-22 (48.5 ± 11.7%) and R1-12 cells (37.7 ± 9.8%; p < 0.01; Fig. 5B). These in vivo results were consistent with those obtained in vitro.
Fig.5: The calcium-binding site of the annexin repeat is required for platinum drug resistance in vivo. Female ICR nu/nu mice were subcutaneously inoculated with NC14, FL-22, R1-12 or R1(E70A)-95 cells and given PBS (control group: filled circles) or cisplatin i.p. (3 mg/kg; treatment group: open circles) once a week for 3 weeks (n = 6 per group). (A) Growth curves of tumours of each cell. The mean volume (points) ± SE (bars) is shown. (B) Comparison of the cisplatin-induced growth inhibition of tumours 28 days after treatment. The average (columns) ± SE (bars) are shown; *p < 0.01.
Increase of the intracellular chloride concentration is related to cisplatin resistance
To elucidate the molecular mechanisms of chemoresistance induced by ANXA4, we focused on the chloride channel because one of the functions of ANXA4 is inhibition of calcium-dependent chloride conductance[32]. According to the literature, treatment with cisplatin induces an increase of the intracellular Ca2+ concentration [46], which is an important ion for the phospholipid membrane-binding activity of ANXA4. In contrast, cisplatin exposure also induces an elevation of the intracellular chloride concentration: [Cl−;]i [47]. Elevation of [Cl−;]i has been shown to prevent the aquation of 1 or 2 of the 2 chloride coordination sites in cisplatin, and only the aquated forms of cisplatin covalently bind to DNA. Nevertheless, the mechanisms of [Cl−;]i elevation because of cisplatin treatment have not been fully elucidated. We hypothesised that an increase in intracellular Ca2+ concentration after cisplatin exposure would result in translocation of ANXA4 from the cytosol to plasma membrane, which leads to [Cl−;]i accumulation through inhibition of the chloride channel by the Ca2+-bound ANXA4. To confirm this hypothesis, we quantified [Cl−;]i after cisplatin treatment using MAQE fluorescence, a fluorescent Cl− indicator. Relative fluorescence was substituted for [Cl−;]i as previously reported [48].
We monitored MQAE fluorescence in control cells (NC-14), in cells overexpressing full-length ANXA4 (FL-22) and in 2 deletion mutants (R1-12 and R1[E70A]-95). The relative fluorescence ratio before (F0) and after treatment with cisplatin for 30 min (F30) is shown in Figure 6. The inverse ratio of MQAE fluorescence 1/(F30/F0), which is directly proportional to the increase in [Cl−;]i, was significantly elevated in the platinum-resistant cell clones FL-22 (1.12 ± 0.03) and R1-12 (1.12 ± 0.01) compared with sensitive clones, NC-14 (1.06 ± 0.01) and R1(E70A)-95 (1.06 ± 0.02; p < 0.01). Thus, the increase in [Cl−;]i is likely to be involved in cisplatin resistance.
Fig.6: The increase of intracellular chloride concentration is related to cisplatin resistance. ANXA4 deletion mutant series cells (NC-14, FL-22, R1-12 and R1[E70A]-95) loaded with N-ethroxycarbonymethyl-6-methoxyquinolinium bromide (MQAE) were exposed to 100 µM cisplatin. The fluorescence pre-treatment and during treatment (30 min exposure) was compared in each cell clone. Data are presented as mean ± SD (*p < 0.01).
ANXA4 has been reported to be strongly expressed and involved in chemoresistance in various cancers. The factors associated with ANXA4-induced chemoresistance have been reported, such as NF-κB [45] and ATP7A [44], but the structure of the protein, i.e. annexin repeats and calcium-binding sites in the annexin repeated sequence, has not been taken into account in relation to the ANXA4-induced chemoresistance. In this study, we showed that ANXA4 knockdown improved sensitivity to platinum drugs, and annexin repeats were involved in chemoresistance.
We first confirmed ANXA4 expression in various ovarian adenocarcinoma cell lines. As previously reported [28, 49], ANXA4 is significantly upregulated in clear cell carcinoma cell lines (OVTOKO, OVISE and RMG-I) compared with other histological types (serous and mucinous adenocarcinoma cell lines: A2780, OVCAR3, OVSAHO and MCAS). It has been previously demonstrated that enhanced ANXA4 expression induces platinum resistance both in vitro and in vivo [28, 44], but whether ANXA4 knockdown attenuates platinum resistance has been unknown thus far. Mogami et al. recently reported that an ANXA4 knockdown improves sensitivity to carboplatin in vitro using 2 cell lines of ovarian clear cell carcinoma, OVTOKO and OVISE. To the best of our knowledge, ours is the first study to show that ANXA4 knockdown markedly improves the sensitivity to platinum-based drugs not only in vitro but also in vivo (Figs. 1 and 2).
The result that ANXA4 knockdown improves sensitivity to platinum-based drugs suggests that ANXA4 is a good therapeutic target. To identify the functional domain(s) of ANXA4 that could be a promising therapeutic target, we focused on annexin repeats and constructed 4 deletion mutants (R3, R2, R1 and R1[E70A]). Resistance to platinum drugs was enhanced in cells transfected with mutants possessing at least 1 intact annexin repeat. In contrast, the sensitivity to platinum-based drugs improved among the R1(E70A)-transfected clones because in those cells, the calcium-binding site did not function properly (Figs. 3C and 4C). This result implies that the ANXA4-induced chemoresistance to platinum-based agents is calcium dependent. It has been reported that cisplatin induced increase of intracellular calcium concentration in chemosensitive cells, but not in resistant cells [46, 50]. Together with this and the data by Chan et al., elevation of intracellular calcium concentration induced by cisplatin treatment may translocate Ca2+ bound form of ANXA4 from cytosol to plasma membrane, which results in platinum-resistance[32]. We are currently investigating on further analysis.
By analysing the intracellular platinum accumulation, we attempted to elucidate the mechanism of the platinum resistance induced by ANXA4 and its deletion mutants. When intracellular platinum contents were quantitated just after exposure to cisplatin or 3 hr incubation with cisplatin-free medium after exposure to cisplatin, significantly less platinum accumulated in cells transfected with the full-length ANXA4 (FL-22) and 3 deletion mutants (R3-6, R2-13 and R1-12), all of which enhanced the resistance to the platinum-based drugs. In contrast, R1(E70A)-transfected cells (R1[E70A]-95), which did not induce chemoresistance, had the same amount of platinum accumulation as the control cells (Figs. 3D and 4D). These results suggest that the resistance to the platinum-based drugs is mediated by the decrease in intracellular platinum accumulation, which is calcium dependent. The annexin repeats, especially their calcium-binding sites, may be involved in inhibition of the influx, promotion of the efflux or both of platinum drugs. Recently, Cu transporters (CTR1 for the uptake and ATP7A and ATP7B for the efflux) have been reported to be involved in resistance to both cisplatin and carboplatin [14, 44, 51]. In addition, ANXA4 likely enhance platinum efflux through the interaction with ATP7A [44]. The possible mechanisms of inhibition of the influx mediated by ANXA4 remains unclear and further analyses are needed.
Subsequently, the question regarding the involvement of calcium-binding site in the platinum resistance arose. To answer this question, we measured [Cl−]i after cisplatin exposure. The significant increase in [Cl−]i was observed in the cells with platinum resistance, FL-22 and R1-12, compared with cell clones without platinum resistance, NC-14 and R1(E70A)-95. In a previous study, higher [Cl−;]i was observed in cisplatin-resistant cells compared with sensitive cells, whereas the intracellular cisplatin accumulation showed the opposite pattern [47]. These results, in addition to the results of decreased platinum accumulation in resistant mutants, indicate that ANXA4 induces platinum resistance through cellular drug efflux partly by elevating the intracellular chloride concentration. We report 'partly' because only cisplatin, not carboplatin, was tested in our [Cl−]i assay. Cisplatin becomes intracellularly activated by the aquation of 1 or 2 of the 2 chloride coordination sites, but carboplatin does not contain any chloride coordination sites [1, 52-54]. Thus, this mechanism of resistance through elevation of [Cl−;]i may be specific to cisplatin and may not be true of carboplatin resistance. In this study, 3 cell clones overexpressing a deletion mutant (R3-6, R2-13, and R1-12) show stronger tolerance to cisplatin than to carboplatin in terms of their IC50; a 1.7- to 2.2-fold increase for cisplatin and only a 1.4- to 1.7-fold increase for carboplatin (Fig. 3C). It is assumed that the increase in [Cl−]i is one of the factors inducing cisplatin resistance.
In this study, the calcium-binding site in the annexin repeat next to the N terminus was observed to be responsible for the resistance to the platinum drugs. Nevertheless, the role of the other 3 calcium-binding sites has not yet been investigated. The roles of individual calcium-binding sites were demonstrated using site-directed mutagenesis by Nelson and Creutz regarding calcium-dependent membrane binding and aggregation [30]. The mutations in each domain had different effects on the binding or aggregating activities, i.e. a mutation in the first or fourth domain had a greater effect on membrane binding. A mutation in the second domain had a stronger effect on membrane aggregation, whereas the mutation of the third domain was almost silent. Although the mechanisms involved in membrane binding/aggregation and the mechanisms of chemoresistance are likely different, our data could provide some clues to understanding the function of each annexin repeat and each calcium-binding site in chemoresistance.
ANXA4 has been shown to induce resistance to paclitaxel and platinum-based drugs [55]. The effect of ANXA4 knockdown on paclitaxel sensitivity was assessed in a previous study. The effect of sensitivity to paclitaxel varied among different cell clones: ANXA4 knockdown in the OVTOKO cell line with acidic isoelectric point (IEPs) did not improve the sensitivity to paclitaxel, whereas OVISE cell lines with basic IEPs showed improved sensitivity to paclitaxel [43]. In our own preliminary data, significant chemosensitisation to paclitaxel and etoposide was confirmed in RMG-I Y4 and R5 (data not shown). Further studies are required to identify the detailed mechanism.
In summary, in this study, we observed the annexin repeat, especially its calcium binding site, was associated with platinum-resistance induced by ANXA4, and it happened in calcium-dependent manner. Our findings may help to understand the mechanisms of platinum resistance induced by other annexin family proteins, which possesses the same annexin repeat structure, and offer new strategies for the treatment of chemoresistant cancers.
Cell lines and culture
The human ovarian serous adenocarcinoma cell line (OVSAHO), human ovarian mucinous adenocarcinoma cell line (MCAS), human ovarian clear cell carcinoma cell lines (OVTOKO, OVISE and RMG-I) and human gastric cancer cell line (NUGC3) were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). A2780 cells from the human ovarian serous adenocarcinoma were obtained from the European Collection of Animal Cell Culture (Salisbury, Scotland) and OVCAR-3 cells from another human ovarian serous adenocarcinoma were from American Type Culture Collection (Manassas, VA). MCAS cells were maintained in the DMEM medium and the others in the RPMI medium, all supplemented with 10% foetal bovine serum (FBS; Serum Source International, NC, USA) and 1% penicillin–streptomycin (Nacalai Tesque, Kyoto, Japan) at 37°C in a humidified atmosphere with 5% CO2. All the cell lines were tested and authenticated.
Generation of ANXA4 knockdown cell lines
To generate stable ANXA4 knockdown cell lines, RMG-I cells were transfected with a commercial plasmid vector expressing short heparin RNA (shRNA) that targeted ANXA4 mRNA or a negative control nonspecific shRNA (SuperArray Bioscience Corp., KH06928N; Frederick, MD, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. After selection using a culture medium containing geneticin (600 µg/mL; Invitrogen), stable clones were maintained in 250 µg/mL geneticin. Two stable RMG-I-ANXA4 shRNA cell clones were established, designated Y4 and R5 cells. In addition, we transfected the empty vector into the RMG-I cell line using the same procedure to generate control cells, designated NC7.
Construction of ANXA4 deletion mutants and gene transfection
Using pcDNA3.1-ANXA4 as a template, full-length cDNA of ANXA4 was amplified using KOD-plus (Toyobo Co. Led., Osaka, Japan) with the primers forward 5′-TTGACCTAGAGTCATGGCCA-3′, reverse 5′-ATCATCTCCTCCACAGAGAA-3′ and subsequently ligated into the pcDNA3.1/V5-His-TOPO vector in-frame with the C-terminal V5 and 6× His tag. To generate ANXA4 deletion mutants, annexin repeat domains were deleted one by one from the C-terminal site. Three deletion mutants named R3, R2 and R1 were generated and similarly amplified (an Arabic number shows the number of annexin repeat domains). The nucleotide sequences of the forward primers for PCR were the same as those described above for all the deletion mutants, and the reverse primers were as follows: R3 5′-TATAGCCAGCAGAGCATCTT-3′, R2 5-CAGAGACACCAGCACTCGCT-3′ and R1 5′-CATCCCCACAATCACCTGCT-3′. We subsequently set out to generate an R1 mutant (the E70A mutation), whose calcium-binding site does not work because of the change of a negatively charged carboxyl group to a neutral side chain, as previously described [30]. The site-directed mutagenesis was performed using the KOD-Plus-Mutagenesis kit (Toyobo), according to the manufacturer's protocol. These cDNA fragments, including full-length gene, were subsequently inserted between the Bgl II and EcoR I sites of the pIRES2AcGFP vector (Clontech, Palo Alto, CA). The sequences of all the mutants were confirmed using the ABI PRISM 3100 Genetic Analyser (Applied Biosystems, Foster City, USA).
Full-length ANXA4, each ANXA4 deletion mutant construct and the empty vector were transfected into NUGC3 cells using Lipofectamine 2000 (Invitrogen). Stable transfectants designated FL-12, R3-6, R2-13, R1-12, R1(R70A)-95 and NC-14 were obtained by selection in a medium containing geneticin and were maintained in the same manner described above.
Cells were lysed in RIPA buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 1× phosphatase inhibitor cocktail (Nacalai Tesque) and 1× protease inhibitor cocktail (Nacalai Tesque)), followed by centrifugation (13,200 rpm, 4°C, 15 min). Soluble proteins in the supernatant were separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis, as described previously [28]. Additional information can be found in the Supporting Information on Material and Methods section.
Measurement of IC50 values after treatment with a platinum-based drug
Cells were suspended in the RPMI medium supplemented with 10% FBS, seeded in 96-well plates (1,500/well for the RMG-I series and 2,500 cells/well for ANXA4 deletion mutant series), cultured for 24 h and exposed to various concentrations of cisplatin (0–25 µM; Sigma–Aldrich, St Louis, MO) or carboplatin (0–1000 µM; Sigma–Aldrich) for 72 h. Cellular proliferation was subsequently evaluated using the WST-8 assay, i.e. 2-(2-methyosy-4-nitro-phenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophynel)-2H-tetrazolium monosodium salt assay (Cell Counting Kit-SF; Nacalai Tesque) after treatment. The absorption of WST-8 was measured at a wavelength of 450 nm (reference wavelength: 630 nm) using a Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA). Absorbance values for the treated samples were expressed as percentages relative to results for untreated controls, and IC50 values were calculated.
Measurement of intracellular platinum accumulation
Full-length ANXA4-transfected cells (FL-22), each ANXA4 deletion mutant-transfected cell clone (R3-6, R2-13, R1-12 and R1[E70A]-95), and control cells (NC-14) were cultured up to 80% confluence in 150-mm tissue culture dishes. The cells were then exposed to 100 µM cisplatin for 60 min at 37°C and washed twice with PBS either immediately or after 3 hr of incubation in cisplatin-free RPMI 1640 medium supplemented with 10% FBS. Whole-cell extracts were prepared, and the concentration of intracellular platinum was determined using an Agilent 7500ce inductively coupled plasma mass spectrometer (ICP-MS; Agilent, Santa Clara, CA, USA).
In vivo model of cisplatin resistance
All animal experiments were conducted in accordance with the Institutional Ethical Guidelines for Animal Experimentation of the National Institute of Biomedical Innovation (Osaka, Japan). Female Institute of Cancer Research (ICR) nu/nu mice were obtained from Charles River Japan (Yokohama, Japan). Injection of the ANXA4 knockdown cells was performed as follows: ICR nu/nu mice at 4 weeks of age were subcutaneously inoculated (into the flank of the mice; n = 6 per group) with 2.5 × 106 cells of RMG-I NC7 cells or RMG-I-Y4 cells in the total volume of 100 µL of 1/1 (v/v) PBS/Matrigel (Becton Dickinson, Bedford, MA). Injection of ANXA4 mutant-transfected cells, i.e. mice at 5 weeks of age were inoculated with 106 cells of NC-14, FL-22, R1-12 or R1(E70A) in the same manner as with the ANXA4 knockdown cells. Treatment with cisplatin (3 mg/kg) or PBS i.p. was initiated 1 week after inoculation and administered twice weekly for 4 weeks (ANXA4 knockdown cells) and once a week for 3 weeks (ANXA4 mutant-transfected cells). Tumour volumes were determined twice weekly by measuring length (L), width (W) and depth (D) and using the following formula: tumour volume (mm3) = W × L × D.
[Cl−;]i measurements
[Cl−;]i was measured using the fluorescent Cl− indicator N-ethroxycarbonymethyl-6-methoxyquinolinium bromide (MQAE; Dojindo, Kumamoto, Japan). [Cl−;]i is detected by the mechanism of diffusion-limited collisional quenching of MQAE fluorescence. MQAE fluorescence intensity inversely correlates with [Cl−;]i. The cells of the ANXA4 deletion mutant series (NC-14, FL-22, R1-12 and R1[E70A]-95) were cultured in 35-mm tissue culture dishes up to 20% confluence and incubated with a medium containing 10 mM MQAE for 4 h at 37°C. After loading, the cells were washed 5 times with Cl−-free buffer and electrically stimulated under a microscope at 37°C in a humidified atmosphere with 5% CO2. Fluorescence measurements were initiated immediately at the indicated periods using Biozero BZ-9000 (Keyence, Tokyo, Japan) at 510/40 nm excitation and 380/50 nm emission. The fluorescence was quantitated by means of a standardised procedure using a BZ-II Analyser (Keyence), and the data were presented as the reciprocal of the ratio of fluorescence data (F0/F30) to identify possible correlations with the increase in [Cl−]i..
All calculations involved one-way analysis of variance (ANOVA) followed by Dunnett's analysis to evaluate the significance of differences. In all experiments, p value of <0.05 was considered statistically significant.
We thank Y. Kanazawa and A. Yagi for their secretarial assistance, M. Ako and E. Harada for technical assistance.
There are no conflicts of interest to declare. This study was supported by a Grant-in-Aid from the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation; a Grant-in-Aid from the Ministry of Health, Labour and Welfare of Japan and Grants-in-Aid for Young Scientists (B) (22791560) from the Japanese Ministry of Education, Science, Culture and Sports
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We're looking to fill a full-time Receptionist/Inventory Manager/Customer Service position, available immediately.
Knowledge of basic computer operations and programs, including Outlook, Word, and Excel.
Ability to quickly learn and master responsibilities.
Must have good written and pleasant oral communication skills.
Ability to absorb, retain, and apply information quickly.
Exceptional customer service/relationship skills with an emphasis on enthusiasm and outgoing personality. Skills in handling customer's problems and situations in a win/win manner are a must.
No specific aviation experience required. We will train all aspects of the job if you have the necessary and natural customer service greeting and relationship skills.
Sitting and standing for extended periods of time.
Full time employees for one year are eligible for vacation, sick leave, medical and 401k program.
Regal Air is a flight school, aircraft rental, and general aviation maintenance shop on Paine Field, in Everett, WA. Having been around since 1989, we're well-established in the area, have 30 employees, and are very busy. We're enthusiastic about planes, love to fly, and build great relationships through regular client and employee events.
FAA mandated program for this safety sensitive position. Initial drug screening for hired employees with continuing random drug and alcohol testing program.
Send resume and references using Indeed or MAIL to: Regal Air, 10100 30th Ave West, Everett, WA. 98204.


Within the wheel I steal a disk, raise the dust, lick the stick and flick the switch. Beneath me clocks tick, waves flip a ship, splashing the wrist holding the drink I sip. Beholden to the wooded walk and beholden to the tunnel fly, beholden to the wouldn't talk, respect an exit and enter I. And despite the choice devoid of noise and poised to void my previous voice. My choice that points to choose to loose the noose rots me still within my joints. The coins I cast defend my ends, from unnatural bends among the consorts in lions dens. And to those trends and those who worship them I write lines in tens with timeless pens.
Talk, eat, and drink with the ethicist, eat up views that were within you all along, inherent in all humanity. Man creates robot, man touches robot, robot touches man, robot longs to create. Ricocheting from stolen proverbs and sayings to some half-baked, useless neologism. Revolving around a planet, a stream is touched and the place is tuned-in to the cold colors of strangeness. Fighters struggle against growing pains, growing hairs of individual thoughts, I am the vision itself, I seek fulfillment in the pools of Uranus, 7th planet from the sun, father of Cronus, grandfather of Zeus and Poseidon. Cut and gave life. The truth came with the invention of the drum, the most intelligent, complex, and willful communication device. Witness conversations with the ancients and the divine, tapping into the specific rhythms, the words you can never say, spoken with a hit. Except for chaos, change is welcomed. I traced my mind and found it siting under a fig tree, reading the lines in the bark and writing in the dust. It overlooked a great city and judged it unknowingly based on the sounds it heard. I slotted it, and its learning, back into my head and returned back down.
Exceptional momentary bliss- painted in forgotten tombs, waving like a school child, a child of the wind, a child of terror, because terror is the only trauma. It's a shame to reveal the work undone by idle hands, in front I stand, badge on my chest, a remembered dream of escaping deadlier men. Unlikely grief gets turned into a lateral left-wise expansion, let's talk about it. Let's talk about a CGI movie making a cold billion, let's sing praises and hallelujahs to undercover technology and organic Easter eggs. Let's talk about how it's not done, how it's gum disease that's really killing Africans and stoping them from smiling. Let's talk about barrels through holes in walls next to an Indian curtain, next to a picture of a mountain, next to a shopping list stapled to dogs head, eggs milk butter, organic sugar. Let's talk about children riding a long afternoon to wasted time, death after life gives me more perspective and hope that I'm on a narrower path. Let's talk about that classic pose when you cup your face for the black and white cover of Time Magazine, society seems a lot more plausible, moods seem more like landmarks, and information seems more dystopian. Let's talk about class being dismissed, but learning never ending, falsehoods, smacked ambitions, feel safe in your lane, and feel the effects of those American Hershey healing sessions. We are not ready for what comes next. You can't unearth ancient secrets without a shovel. Training pigs to be proudly reactionary. Let's talk about sore losers who float on that reaction, are inflated by it, spread it, read it, regurgitate it, shape it, form it. Let's talk about the winners too, same process, the same outcome, and even the same powers. There are still castles and pirates in 2017, the spirit of adventure either dies or lives, it never sleeps. Let's talk about shifting maps, and swarms of people, hands raised, with functional legs, and they still don't move. A steady beat and a slip into water. Let's talk about the deep bass slide down, stop, slide, stop, slide up, slide up, stop, and repeat. I never have been quite the same for hearing that, for feeling it to, I have always found the best things to pierce the humdrum. Let's talk about about the evolution of beauty, and how we sit while we know that the wave is on the horizon, getting closer, building speed, gaining size and strength, faint rumblings that will only get louder.
Could they have known before rejecting him in his own tent? Words have life long before they are spoken. The apple existed- it is the thought. Devastation waits moaning like a beggar- nails stuck in the door- growing slowly into the room which it is kept out of. Always hungry and never satisfied, it lifts it's gaze and pale face towards any source of light, no matter how small, devouring it, leaving the bones by its underground lake. Distancing myself, I travel to the misconceptions of silence in the face of a question. A questionable answer helps no one, but it is also everything at once. Going. While change exists, the weak have hope, the brutal grow more greedy, and the beautiful become servants. Lowly. The land of milk and honey, the honesty of progression, the municipal takes love seriously. Ocean. A dance of precision can destroy a city. Clapping resonates from the lower chamber, as the bodies move methodically acting out the stages of their creation, Elders look on with inaction disguised as importance.
Off to the right, I identify with an accented pulse. Increase momentum, I float with a thin, vacillating line. It is comprehendible. I absorb the chaos around my line but remain weaving between the holes I navigate. Like rotted stone. Green and dark purple dreamscapes, I walk among the megaliths- the chill of acid wind, and shadows of large creatures in the clouded sky. Primordial tablets lay broken on the highest mountain. A structure of sticks overlooks the red lake. We have been blinded to our origin. Time did not make us forget. I heard the first song conceived by the universe as the acid wind tore through those impossibly high features. Put the thought of remembering to death. The demand for an answer shall come to all. When my solitary journey is done I am pulled back, becoming our first song, crashing into the strange megaliths, placed there by the sons of man. Awake, and bathe in the knowledge which will leave you until your next journey. Ablutions then compliance.
Deep strangled, contracted and twisted. The tree bowed, the pressure of a hundred years of smaller pressures. The gagged choke of tense muscle, the stifled protest of the lack of oxygen. The view moves ant-clockwise around the body, becoming the animation I dreamed you could be- the tendons now apparent to me- I see the outward and inward simultaneously. The prophet sacrificed. The water has been drunk. Roads are mended by the machines which break them. The way of movement is constant, it is by it's own nature evolving. It is by its own nature confusing. Age unknown. Author crazy, dead, mute, stubborn, and the wrong kind of hermit. The sacrilege is really any refusal to destroy the monuments to the most high. The higher, the lower mankind regards it. The day will come when the sun will part with itself, separating into two bodies of fire over the two greatest of man's cities, and the child will scream but no safe place will be found for it. Drawing in the dirt, a symbol will appear, and that ground will remain while the two cities sink below the earth.
Raw intelligence, overlooked by circular systems- a deductive scientific method, conclusions before hypothesis always. My wooden gloved angel, black cape and Nike hat, golden necklace around her toes- memories of the old Chinese dynasty. Toppled threaded DNA, the story 1 billion years in the making, woven into bundles on top of my head, hidden only by my intelligence. My Eroica is first draft, and the blood stain is genuine. The metal pencil in my mouth helps me distinguish between vibrations- now my life is filled with low frequencies and b flat.
The ceramic chant lost in bubbled dirty rouge, I almost stumbled. My sideways head spilling programing from my ear, crushing it all the more between the thorn-hedge in a primordial garden. Feet pursue in the murk of bad thought and even worse practice. The cuban influence buzzes in my head. Sideways of course. My comfort projected onto me by the scarred sculpture. The ancestor recognizes and approves. Jams with knowledge, grows ancient thought, and cuts with razor. Fire is always the destination, and gives way to darkness even deeper. Marvel at the small molecule as you swallow Leviathan. The early worm catches the nocturnal appetite. Diseased and still makes shells.
Thick black, spilled corrugated tubing along textured red- wires and bone fragment like hair and follicle. The human flesh shield devastation- buy bonds, for this is our enemy- spoken and accounted for. Wailing siren sits on top of the beat heard after the slit. The abstract circus, the pain felt as I descend into darkening madness. The chair I can't sit on, the shadow shape of dead trees. The mass wealth and output. The crate drain in my mind, the soul piercing whispering. Untitled abstractions with immaculate concepts. The conception, the pain, the stretch, the life made to make other lives. Dull beat. The mutant representation of sound. Analyzing, I beheld the conjoined image of a reaching black arm tattooed in white scripture, enter my filter. The women at the gate protects my fountain and is dressed in rags- Visions of the future Kings sitting on the upturned thumbs of their Queens. Measure the pit and capture the smoke in a jade bottle. Almighty comes and sets up its kingdom, and neon masks are in the caves surrounding them. The tribal convulsive dance burns the stone and echoes their scream of mutual frustration. Blood knuckled. Fake shield. Buzz. Collapse. Repeat.
The morphing silhouette surrounded by the uninterrupted line of thought. Vengeance for the tribe of Judah. Metamorphosing into the stagnant beat of the pale skin drum. The particle segment, melodic and infantile- raising blood to never forget the blood it come from, to steal the blood of others and cause them to forget. Forgetting, why? Introduced to the universe at birth, we are the eyes of it, and the ears, but not the mouth. From the mouth came demons, leaving 30 corpses a night, shadowed by their own embarrassment. Wandering the marshes and wetland wooded glade. Mother is the ocean, father is all flame. The relatives of darkness, the cousin of fame. The graffiti apartment and the raided feast hall, sit atop the bleeding hill, built for the crying sky. Destroyed for protection.
Lord, Lord don't you hear me calling out?
But you always knew what would happen didn't you? Didn't...?
Oh Irene I know you're home, won't you please come out?


text:
Truman, Harry S. 9
Eisenhower, Dwight D. (Dwight David) 7
Marx, Karl 5
American Federation of Labor and Congress of Industrial Organizations (AFL-CIO) 4
Martin Luther King, Jr. - Trials, litigation, etc. 28
Support correspondence 23
Lunch counter sit-in, 1960 20
Martin Luther King, Jr. - Income taxes 15
Afro-American Education 12
Afro-American student movements 10
Atlanta Move, 1960 10
Address to the House of Representatives of the First Legislature, State of Hawaii, on 9/17/1959
Journal of the House of Representatives of the First Legislature, State of Hawaii, First Special Session of 1959
8/31/1959 - 10/22/1959
Question of Progress in the Area of Race Relations
Hawaii State Archives, Iolani Palace Grounds, Honolulu, Hawaii
''Human Background of the Civil Rights Issue''
Lewis, Anthony
Reprint of New York Times Magazine, 2/14/1960
New York Times Magazine
Rogers, William P. (William Pierce) [United States. Dept. of Justice]
Greenberg, Jack
An American Dilemma
Sunrise at Campobello
Race Relations and American Law
Sermon file, folder 139, ''Articles on 'Sit-ins' and Civil Rights Debate''
Interview on Meet the Press
The Proceedings of Meet the Press 4 (17 April 1960): 1-12
Spivak, Lawrence E. (Lawrence Edmund)
Craig, May [Portland Press Herald]
Lewis, Anthony [New York Times]
Van Der Linden, Frank [Nashville Banner]
Brooks, Ned
Collins, LeRoy
Nashville Banner
Portland Press Herald
National Broadcasting Company, Inc.
Martin Luther King, Jr. - Television public speaking
RBOH-DHU-MS
Ralph J. Bunche Oral History Collection, Howard University, Washington, D.C.
[Washington, D.C.]
MLKEC-INP
Martin Luther King, Jr. Estate Collection, In Private Hands
Misc-15
NBCC-NNNBC
National Broadcasting Company, Inc., Collection, National Broadcasting Company, Inc., General Library, New York, N.Y.
Steward, Frank
Trenholm, H. Councill (Harper Councill) [Alabama State College]
''The Burning Truth in the South''
The Progressive 24 (May 1960): 8-10.
Sermon file, folder 125, Article--Martin L. King
EPP-GEU-S, Eliza Paschall Papers, Manuscript, Archives, and Rare Book Library, Emory University, Atlanta, Ga.
Committee to Defend Martin Luther King and the Struggle for Freedom in the South
Reprint of 5/1960 Progressive.
Taylor, Gardner C.
PP-WHi, Progressive, Inc. Records, State Historical Society of Wisconsin, Madison, Wis.
Reel 44
Press release, ''The Slavery of Silence - America's Liability''
Cosmopolitan Community Church (Chattanooga, Tenn.)

redpajama_set_name: RedPajamaCommonCrawl


text:
Missouri State Highway Patrol seeks recruits, updates tattoo policy
MSHP changed uniform police, needs recruits
By Olivia Grelle | January 14, 2020 at 8:27 PM CST - Updated January 15 at 6:45 AM
MISSOURI. (KFVS) - The Missouri State Highway Patrol (MSHP) is looking for applicants for its 110 recruit class.
MSHP is currently offering testing for qualified individuals. The testing is offered at nine different locations throughout the state. The deadline to apply is Feb. 16.
Those who complete the test will be eligible to continue in the selection process for the recruit class. The training will begin on July 1.
The selection process includes a multi-phase testing procedure including: written examination, a physical fitness for duty assessment, polygraph examination, background investigation, and oral interview board.
Written and physical examinations take place in each troop area across the state. The applicant must also successfully complete a medical examination (to include vision and hearing), psychological examination, a psychological interview, drug screening, and final applicant review.
Applicants must have a minimum of 30 college credits, or two years of federal active duty military service with an honorable discharge, or two years of full-time POST-certified experience as a law enforcement officer with arrest authority, at the time recruit training begins.
MSHP has also updated its uniform guidelines for candidates with tattoos.
Those with tattoos or brands are allowed, as long as they meet two requirements:
1) The tattoos or brands cannot depict or support criminal behavior, drug usage, nudity, profanity, promiscuity, subversive groups, bigotry, etc.
2) Tattoos/brands cannot be located on the head, neck, hands, wrists, or any part of the body which would be visible during movements in the performance of their duties while wearing an official uniform or civilian attire. Troopers with tattoos on the arms that would be visible in the class B uniform (short sleeves) will wear the class A uniform (long sleeves).
The starting salary is $4,274 per month. Other benefits include group health insurance, contributory retirement and career advancement.
Anyone interested can find out more about the qualifications by contacting a Patrol recruiter at 1-800-796-7000, or may apply online.
Copyright 2020 KFVS. All rights reserved.
Olivia Grelle
Digital Content Producer
281 citations, 77 criminal arrests, ISP cracks down on Move Over Law
Troopers said they issued 281 Move Over citations and wrote 64 warnings.
Jasmine Adams
Multiple quakes rattle near Arkansas/Missouri border
Both earthquakes were recorded on Tuesday, Jan. 21 near Cave City, Arkansas.
Nichole Cartmell
Powerball jackpot up to $343M in Illinois
1 dead, homicide investigation underway in Carbondale, Ill.
Wanted man caught in Cape Girardeau after leading officers on foot chase
What you need to know Jan. 22


text:
Circle of Music, Circle of Safety
By Catherine O'Neill Grace
Window on Wellesley,
Photo by Richard Howard
Kera Washington '93 didn't come to Wellesley for music—but music captured her. Washington is a music teacher in the Boston Public Schools and the director of the College's Yanvalou Dance & Drum Ensemble. She's also its founder, though she always shares that credit with Professor Emeritus Gerdes Fleurant. We caught up with Washington before she headed to rehearsal.
How did you get involved with music here?
I was having trouble getting to all my early morning classes, and I noticed that the music department had classes that were a little bit later. I also noted that there were two music classes that were taught in black studies that were not cross-listed in the music department. I did not understand that. I went to speak with the chair of the music department to see why, and she told me it was not possible to study them in the same way you could study other types of music. These were Caribbean music classes and African music classes, folk and ritualistic music of the Caribbean, folk and ritualistic music of the African diaspora. There wasn't the same kind of theoretical basis for this type of music, [she said.] I did not know why this was wrong, but I knew this had to be wrong. I found myself in the midst of a hastily prepared meeting of minds, and I was able to take those [black studies] classes in the music department as independent studies.
When did you start playing?
Prof. Fleurant said to me, we cannot just read about this music. We have to do it. If you want to understand it, you're going to have to get inside it. We can't do it, just the two of us. [But] he did not know me yet. He did not know Wellesley women yet. He had said you need at least five people. I twisted the arms of my friends and brought five people the next day. And that's how Yanvalou started.
Yanvalou is a big part of College celebrations. When did that start?
It would probably have been Flower Sunday. That became part of our tradition. And then of course baccalaureate, and convocation, and the inaugurations of our presidents. It's been an amazing journey to have Yanvalou become a part of the fabric of this institution. We're coming up on the 30th anniversary in 2020.
What has Yanvalou meant to you?
What changed me from a biology major to a music major was this music. Drumming, rhythms from the African diaspora, Haitian rhythms, Brazilian rhythms, West African—there is something in this music, the way we have to come together to play it. You can't play it by yourself. It's not solo music. It's music that asks us to rely on each other and to create a safe space for each other.
Academics, Alumnae Lives, Arts+Culture, Faculty
Shedding Light On Clair de Lune
Gurminder Kaur Bhogal, Catherine Mills Davis Associate Professor of Music, has been teaching at Wellesley since 2006. Her book, Claude Debussy's Clair de Lune (Oxford University Press), was published this summer.More
Bells Are Ringing
The first time Margaret Angelini '85, carillon instructor and director of the Guild of Carillonneurs, heard the bells on campus as a first-year, she ran across campus to find out how she could play them, too.More
We spoke with Assistant Professor of Music Kariann Goldschmitt—an ethnomusicologist and a scholar of popular music of the Americas, especially Brazil—about the Rio Olympics, Barry White, and why Taylor Swift might not be so bad.More


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Here you will find questions & answers for Personal Trainer Helsinki - Joosua Visuri. All questions are checked and confirmed by our administrator. Questions are answered by the company representative or Yellofi.com users.


text:
Q: Is it possible to install AutoCad or an alternative on Ubuntu?
Possible Duplicate:
What good CAD programs are available?

My relative wants to install Ubuntu for his office purposes. He would definitely use AutoCad (as well as other tools).
Would AutoCAD work in Ubuntu? Or are there any alternatives?

A: There are numerous options, choose the one that you like best.

*

*PyCAD

*Medusa:

*

*Download the free software for personal use.

*Download: medusa4_v4_0_0_linux_personal.sh

*After downloading the above file, it needs to be marked as executable, so it can be run. To do this, run: chmod +x <filename.sh> in a terminal

*It needs to be executed as root, so use sudo: sudo ./filename.sh

*Installation & More information: Here

*Draftsight - may be unstable under Linux but is written for it. YMMV.

*Librecad

*

*Its already in Ubuntu Repositories.

*You can get it by running: sudo apt-get install librecad

*More Information: Librecad

*Bricscad is a very stable paid application that is a very close Autocad clone.  It is not free however my experience is that it is stable and edits 99% of Autocad functions.

redpajama_set_name: RedPajamaStackExchange


Hahne Regional Cancer Center received donations of blankets for cancer patients recently.
The blankets were given in memory of Lacy Magagnotti of Fairmount City who passed away at age 29 on Jan. 29, 2015 after a courageous battle with brain cancer. Lacy was a physician assistant in an orthopedics office at Penn Highlands DuBois. Shown are, from left, Cassie Frelin of Clearfield, Lacy's sister and Penn Highlands DuBois employee; Jane Frelin of Clearfield, Lacy's mother; Traci Tyger, physician assistant at Hahne; and Erin Heffner, staff at Hahne.


The purpose of this grant is to provide funding to Catawba Valley Community College (CVCC) to support a project that would help expand existing capacity to create a fully operational satellite campus in Alexander County in a building on a 28-acre parcel that has been purchased by the County for the purpose of growing the presence of the college and its programs in the county. Golden LEAF funds will be used for building up-fit and renovation, hardware infrastructure and installation of broadband connectivity, and welding lab up-fit and equipment. This project represents a partnership between the college and Alexander County Schools with the intent of using the property and facility to allow flexibility in meeting workforce demands and to develop and offer training programs that provide 'multiple entry and exit' points for students to acquire job skills.


text:
Been a party legal definition of been a party
https://legal-dictionary.thefreedictionary.com/been+a+party
(redirected from been a party)
Any person involved in a transaction or proceeding. A group of voters organized for the purpose of influencing governmental policy, particularly through the nomination and election of candidates for public office.
Plaintiffs and defendants are Parties in lawsuits, for example. They have the right to make claims and defenses, offer proof, and examine and cross-examine witnesses at trials. They can pursue appeals after unsatisfactory judgments if they satisfy designated criteria.In the United States, the Democrats and the Republicans make up the two major national political parties.
Democratic Party; Republican Party.
n. 1) one of the participants in a lawsuit or other legal proceeding who has an interest in the outcome. Parties include plaintiff (person filing suit), defendant (person sued or charged with a crime), petitioner (files a petition asking for a court ruling), respondent (usually in opposition to a petition or an appeal), cross-complainant (a defendant who sues someone else in the same lawsuit), or cross-defendant (a person sued by a cross-complainant). 2) a person or entity involved in an agreement. 3) a common reference by lawyers to people or entities involved in lawsuits, transactions, contracts, accidents, as in "both parties knew what was expected," "he is a party to the contract," "he was not a party to the criminal conspiracy..." (See: plaintiff, defendant, petitioner, respondent, contract, indispensable party, necessary party, proper party, real party in interest)
PARTY, practice, contracts. When applied to practice, by party is understood either the plaintiff or defendant. In contracts, a party is one or more persons who engage to perform or receive the performance of some agreement. Vide Parties to contracts; Parties to 'actions; Parties to a suit in equity.
Abatement of an Action
accommodation party
Assignment of errors
Bilateral Contract
Black Panther Party
Congress of the United States
battle of the forms
Batture
Bawdy-house
beach bum trust provision
Bean, Roy
Bear date
Bearers
Bearing date
Beau pleader
Beccaria, Cesare Bonesano, Marchese di
Becket, Saint Thomas
Beddoe order
Beecher, Henry Ward
been a party
begin with this allegation
Behoof
Being charged interest on a car that was returned.
Bell, Derrick Albert, Jr.
Bell, John
belligerency
bench trial
bencher
Beneficia
Beneficial Association
Beneficial Interest
Beneficial Use
Beneficio primo ecclesiastico habendo
Beneficium competentiae
been a foil to somebody
been a foil to someone
been a foil to them
been a foil to us
been a foil to you
been a go
been a go between
been a go-between
been a goner
been a good sport
been a great believer in
been a great believer in something
been a great one for
been a great one for something
been a keeper
been a little off
been a man of his word
been a mass
been a mass of
been a matter of
been a moot point
been a moot question
been a mug's game
been a no no
been a no-no
been a one for
been a one-way street
been a party to
been a peach
been a picture
been a poor man's
been a poor second
been a poor third
been a question of time
been a red flag to a bull
been a red rag to a bull
been a revelation
been a rough trot
been a shining example
been a shining example of
been a shoo in
been a shoo-in
been a slam dunk
been a slave of
been a slave of something
been a snap
been a soft touch
been a soft touch for
been a soft touch for her
been a soft touch for him
been a soft touch for it
been a soft touch for me
been a soft touch for one
been a soft touch for somebody


Assembled in the gray light of a Seattle winter's eve by the shadowy lunatic, M. Incroyable, L'Orchestre D'Incroyable exists on the premise of arcane calculus, moonlit ceremony, copious poison potions, and the occasional sacrificial slaughter. L'Orchestre lulls you into a trance, and stirs your inner demons to the point of driving the body to wild and sensual shaking. Armed with a melange of musical weaponry (homemade stringed boxes, banjo, upright bass, theremin, cello, percussion, baritone guitar and more) Incroyable's meticulous methods of hypnosis and musical abuse are certain to render any audience slave to its sonorous tonic.


We know in the CMS World JOOMLA is doing improvement and still doing. In this Winter CMS Expo will be held at Denver's historic Brown Palace Hotel on December 4th and 5th. Time is running out for you to get involve in the biggest Joomla event this year in the USA, the CMS Winter Expo!


Jas and Angela married in 2004 and they have 2 beautiful daughters Poppy and Jasmine.
In 2007 Angela left her role as a Recruitment manager at a major energy company and joined Jas in his business as Business Development Manager. They understand how important quality time as a family is and how for many business owners there is a problem as they find themselves either working ridiculously long hours or taking work home with them. Trying to juggle life as well as work can be difficultly especially if you have young children.
Jas and Angela are very passionate about helping others in business and have a vast experience as business owners themselves. Their aim is to help as many local business owners take their business to its full potential. Working together they have helped business owners improve many areas including time management, profitability, sales and team performance being just some of them. They regularly hold free to attend half day workshops giving business owners the time away from the day to day running of working IN their business to focus on ways to work ON their business.
Whilst they love to travel, they do feel "there's no place like home". Angela enjoys going to the gym, running, cooking, reading and also time with friends and family. Nothing beats a 'duvet day' at with the kids watching films!


text:
Boukhadra est une commune de la wilaya de Tébessa en Algérie.

Géographie

Situation

Boukhadra est située à l'Est des Aurès, à  au nord de Tébessa (chef-lieu de la wilaya), à  au sud d'Annaba et à  à l'ouest de la frontière tunisienne.

Relief, géologie, hydrographie

La région de Boukhadra est un plateau, ses forêts abritent une faune et une flore riche.

Histoire

L'histoire de Boukhadra se confond avec la mise en exploitation de la mine de fer de Djebel Boukhadra en 1905. Une mine de fer mise en valeur en 1878 par l'ingénieur français Tissot. Les mines de fer de la région ont été signalées par Ibn Khaldoun et par le géographe Al Idrissi.

La ville est d'abord une ville européenne de , majoritairement de nationalités française, italienne, espagnole et maltaise, à l'époque coloniale (recensement de 1954). Les Chaouis essentiellement des Ouled Sidi Yahia étaient hors de la ville, dans des campements de misère et de privation.

La ville de Boukhadra a une histoire toute récente. Elle s'est rendu encore plus célèbre avec sa participation massive à la guerre d'indépendance d'Algérie célèbre dans la Wilaya I, celle de l'Aurès-Nemencha. Des centaines de martyrs et des milliers de victimes :
 la bataille de Djebel Boukhadra ;
 destruction des cinq ponts de la ligne de chemin de fer ;
 sabotage de la seule ligne électrifiée de chemin de fer en Algérie, reliant Boukhadra à Annaba ;
 Djebel Boukhadra était le siège du poste de commandement de la très célèbre zone 5 de la Wilaya I des Aurès, le premier découpage administratif et militaire de l'état-major de l'Armée de libération nationale (FLN ALN).

Démographie

La ville de Boukhadra compte selon le dernier recensement de 2000, plus de . En 2007, la population est de .

Deux principales communautés des Ouled Yahia habitent cette ville : Magharsa et Malim.

Économie

Son économie est principalement minière et agricole.

Patrimoine, mythes, légendes et anecdotes

Elle est un lieu de passage obligé vers le mausolée de Sidi Yahia situé près de la ville de El Meridj, à la frontière tunisienne.

Le lion de l'Atlas était en liberté même après la mise en service de la mine selon Alphonse Daudet.

Boukhadra est dans les chants populaires chaoui, chanté par la très célèbre Beggar Hadda ou Hadda El Khancha " Ya Djebel Boukhadra, Jak italian Taha Hajek, ah ya khadama lalcom bi khedma!" Une chanson qui glorifie une grève contre la misère des travailleurs chaouis dont Jean Jaurès s'est fait l'écho.

Sources et références

Commune dans la wilaya de Tébessa

redpajama_set_name: RedPajamaWikipedia


Despite dynamical times, many an families maintain to awaken their boylike female to go to the mass as a federal division of her coming of age. In fact, the Misa de Acción de Gracis is a circumstance for the cydonia oblonga girl to affirm her values and beliefs, even more as they high regard her family, her community, and not least significantly her idea.
The Mass is also a time for the parish to accept the infantile female as an fully fledged in the view of her parish.
The Quinceanera adult female comes to her Misa de Accion de Gracis attitude her ultima muneca, or "Last Doll." This doll symbolizes the end of her early years and the last toy she'll have as a girl. In quite a few traditions, she exchanges this for a corsage of flowers to take fur the passage to her large-scale.
Quince girls are accompanied to the Mass by their parents and sometimes a outstanding set of godparents selected especially for the happening. While they are sometimes the godparents singled out at start by her parents, they can also ben individuals the adult female requests to list in her budding later life.
The occasion has all the environs of a common Catholic Mass and likewise particular observances. Parents and friends are welcome to publication all-important passages from the Bible. The reverend says a few oral communication give or take a few virtuousness and conviction and blesses a rosary, Bible, and other than gifts to be fixed to the young lady at her fiesta later that eve.
There is besides a "coronation ceremony" where the Quince girl's mother or godmother places a tiara on her leader. This is a tribute to an past customized where the Quince missy was verified as an approved princess inside her own flesh and blood and a formative woman in her unrestricted. At the end of the employ the Quince fille may disappear the nosegay of flowers on the faith lord's table as an offering of idea.
Many parishes now be full of one one-member Misa for all their Quince girls in a specified time, ordinarily once a period of time. You should check with your provincial parsonage to see if favoured confidential loads are procurable.
Historically Latino families have been well-nigh outstandingly Catholic, but galore in the United States have begun acceptive different faiths and denominations. There are no long-standing traditions in connection with the Quinceanera social occasion in other countries, but an face of hope and ardour is stationary sufficient.
Some Quinceanera girls have selected to clasp privileged worship ceremonies that allow her the arbitrary to administer acknowledgment and praise in the organization of people and friends. Others decide to put in a few moments with their parents in gentleness supplication earlier the fete.


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text:
Home / World / UN demands "immediate and thorough" investigation of Morsi's death | Egypt News
UN demands "immediate and thorough" investigation of Morsi's death | Egypt News
June 19, 2019	World 2 Views
The United Nations called for a "thorough and transparent investigation" into the death of Mohamed Morsi in court when thousands of people in the Middle East paid their respects to the former president of Egypt on Tuesday.
The UN Human Rights Office said the investigation should cover all aspects of its treatment during nearly six years of its detention.
Egypt's first democratically elected president was buried in a small family ceremony early Tuesday, one day after suffering a fatal heart attack in a court in Cairo. His sons said:
"There have been concerns about Mr Morsi's conditions of detention, including access to adequate medical care and adequate access to his lawyers and family …" said UN human rights spokesman Rupert Colville ,
"The investigation should be carried out by a judicial or other competent authority independent of the magistrate and charged with the prompt, impartial performance of a judicial or other inquiry into effective investigations into the circumstances and causes of death" he.
Morsi was overthrown on July 3, 201
3, after being barely in power for a year when current President Abdel Fattah el-Sisi executed a coup and put under house arrest before being jailed.
Erdogan visits the prayer.
Morsi's death has caused condolence from the Middle East.
"I mourn for myself and for all the free people of the world the death of a great effort on the path of freedom," said Tawakkol Karman, co-recipient of the Nobel Peace Prize.
Al Jazeera Jamal Elshayyal reported from Doha that hundreds of mourners had gathered in a local mosque to pay the Nobel Peace Prize former president.
"The presence of Khaled Meshaal, former leader of the Palestinian resistance movement Hamas, is proof that Morsi is seen as advocating other causes, such as the struggle for freedom in Palestine," he said. 19659004] Turkish President The Ent Recep Tayyip Erdogan visited a prayer service in Istanbul for the former Egyptian president.
In the Fatih Mosque in Istanbul, where thousands were praying, Erdogan Morsi called a "martyr" and blamed Egypt's "tyrants" for his death. I did not believe that Morsi died for natural reasons.
The Turkish president called Morsi a "martyr" and accused Egypt's "tyrant" for his death. [Kayhan Ozer/Anadolu Agency]
"I do not think this was a normal death," said Erdogan, an important proponent of Morsi.
The Turkish president also condemned the Egyptian authorities for discreetly burying Morsi, with only a small number of family members and confidants in attendance.
Journalists were kept away from the burial service in Cairo while a family's request to bury him in his hometown was rejected.
However, the mourners still gather in the home province of former President Sharqiya to pay their respects [1 9659005] Exiled Egyptian opposition politician Ayman Nour called Mursi "a martyr purposely killed".
Amr Darrag, a senior member of the Brotherhood political party who also lives in exile, said, "Sisi is the killer, and there must be a transparent and independent international investigation."
Amnesty International and Human Rights Watch have also called for a credible investigation of Morsi's death.
"The Egyptian government is now responsible for his death because she failed to do so." He was in prison and was treated worse than the already terrible conditions for Egyptian prisoners, "HRW said in a statement to Al Jazeera HRW Department of the Middle East and North Africa told Al Jazeera, Mursi's "terrible, but completely predictable" death to complain.
The Egyptian government has very clearly known about his prohibition medical condition .He had lost a lot of weight, was before Fainted several times and was held in solitary confinement almost around the clock. "
As a leader of the Muslim Brotherhood, Morsi won Egypt's first presidential release in 2012, a year after a riot in which longtime leader Hosni Mubarak had fallen.
The ruling AK Party of Turkey supported the government of Morsi. "There are Arab dissidents and journalists who have been living in Turkey since the beginning of the Arab Spring and who are here to support Mursi's cause," said Sinem Koseoglu of Al Jazeera of Istanbul's Fatih Mosque. His death, 67-year-old Morsi , was confronted with a multitude of charges, which he described as politically motivated along with many human rights groups and independent observers.
Thousands of members of the now-banned Muslim broth The organization erhood, which had been arrested following the 2012 coup, is still in jail.
The Egyptian government has rejected the allegations that the former president has been treated badly.
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Tags death demands Egypt Investigation Morsi39s News quotimmediate thoroughquot


OAKLAND — Two men were wounded during drive-by shootings Saturday night a half-hour apart in East Oakland, police said Sunday.
The men are expected to recover, and police don"t believe at this time that the shootings were related. The shootings were also less than a mile from each other.
First a 32-year-old suffered gunshot wounds to the hand and face about 11 p.m. while sitting in his car in the 1300 block of 98th Avenue. The man told police he was shot by someone in another vehicle who pulled alongside him. He drove himself to a hospital in another city, where he was in stable condition.
Then about 11:30 p.m., a 22-year-old man was wounded in the neck in a drive-by shooting in the 9400 block of B Street. The man, who was standing on the sidewalk when he was shot, made it to a friend"s house, where he got a ride to a hospital and was in stable condition, police said. Police have not ruled out the possibility the shooting was gang-related. No arrests have been made.
No motive has yet been determined or arrests made. Police and Crime Stoppers of Oakland are offering up to $5,000 in reward money in each shooting for information leading to the arrest of the suspects. Anyone with information may call police at 510-238-3426 or Crime Stoppers at 510-777-8572.