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Back to Journals » International Journal of Nanomedicine » Volume 10 » Issue 1
Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice
Authors Wang M , Wu B, Tucker JD , Lu P, Lu Q
Received 5 June 2015
Accepted for publication 8 July 2015
Published 3 September 2015 Volume 2015:10(1) Pages 5635—5646
DOI https://doi.org/10.2147/IJN.S89910
Editor who approved publication: Dr Thomas J Webster
Mingxing Wang, Bo Wu, Jason D Tucker, Peijuan Lu, Qilong Lu
Department of Neurology, McColl-Lockwood Laboratory for Muscular Dystrophy Research, Cannon Research Center, Carolinas Medical Center, Charlotte, NC, USA
Abstract: In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases.
Keywords: cationic polyelectrolytes, antisense delivery, exon-skipping, PMO, muscular dystrophy
Antisense therapy has grown as a powerful strategy for the treatment of genetic disorders and infections. Antisense oligonucleotide-mediated exon-skipping has been demonstrated as a promising therapy to treat Duchenne muscular dystrophy by skipping specific dystrophin gene exon(s) to restore the reading frame of the mutation-containing transcripts.1–12 Antisense oligonucleotides are short, typically 15–30 base pairs of single-stranded sequences of synthetic nucleic acids, or chemically modified analogs, which have the ability to hybridize to specific targets by the base-pairing rules. Of the synthetic oligonucleotide chemistries, phosphorodiamidate morpholino oligomer (PMO) is most widely used for exon-skipping in the dystrophin gene currently being applied in clinical trials.11–15 PMO, as a synthetic mimic of nucleic acid, has deoxyribose rings replaced with morpholino rings and linked through phosphorodiamidate inter-subunits, making it neutral under physiological conditions. It has exhibited excellent stability, and lower toxicity compared with its counterparts, such as 2′-O-methyl-phosphorothioate RNA and peptide nucleic acid.16,17 However, the relatively charge-neutral nature of PMO is associated with poor cellular uptake and rapid clearance from the bloodstream, which constitutes a major obstacle for effective delivery when treating neurogenetic disorders. Studies in a number of animal models have demonstrated that a significant therapeutic effect can be achieved with high doses of PMOs, which could be cost-inhibitive and have an increased risk of toxicity, especially for long-term systemic administration.5 To improve delivery efficiency, PMOs chemically modified with cell-penetrating peptides or dendrimeric octaguanidines have been devised, and are reported to show significant improvement in targeting dystrophin exons, leading to near normal levels of dystrophin expression in body-wide muscles by systemic delivery.3,4,6,9,10 However, the cationic modification is associated with higher toxicity, with LD50 near 100 mg/kg, making it unsuitable for use in clinical applications.3,6 Furthermore, the complicated synthesis and purification in modification increases the costs significantly, and potential immune responses to the targeting peptide could prevent repeated administration. The non-virus-mediated delivery strategy remains attractive because of the vector's structural flexibility chosen from synthetic or natural compounds, capacity for delivery of larger therapeutic agents, ease of handling, greater safety, and less expense than viral vectors,18–20 especially for those commercial available compounds that have been widely applied in the field of environmental biology and biotechnology.
Polyelectrolytes (PEs) are polymers with ionizable groups that dissociate, leaving ions on the polymer chain and counter ions in polar solution. PEs have been investigated for numerous biochemical and medical applications.21,22 The permanent cationic characteristics, such as valence, charge density, and structure, would be expected to have an important impact on the compaction of oligonucleotides and subsequent transfection. In this study, we investigated several commonly used PEs as carriers for delivery of antisense PMO in vitro and in vivo. These include cationic PEs, poly(diallyldimethylammonium chloride) (PDDAC), which is widely used as a flocculant agent and biosensor composite;23–25 poly(acrylamide-co-diallyldimethylammonium chloride) (PADAC), and poly[bis(2-chloroethyl)ether-alt-1,3-bis[3-(dimethylamino)propyl]urea] quaternized (PBEBP), which are used in waste treatment for laundry, emulsion breaking, sludge dewatering and drainage; and protamine sulfate containing cell-penetrate peptide, which has been studied in gene therapy for increasing transduction by both viral and non-viral mediated delivery mechanisms.26,27
Dulbecco's Modified Eagle's Medium (DMEM), penicillin-streptomycin, fetal bovine serum, l-glutamine, and HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer solution (1 M) were purchased from Gibco, Invitrogen Corp (Carlsbad, CA, USA). Phosphorodiamidate morpholino oligomer PMOE50 (5′-AACTTCCTCTTTAACAGAAAAGCATAC-3′), PMOE23 (5′-GGCCAAACCTCGGCTTACCTGAAAT-3′), and Endoporter were purchased from GeneTools (Philomath, OR, USA). PEs, protamine, and all other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), unless otherwise stated.
Cytotoxicity was evaluated in a C2C12E50 cell line using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based assay. Cells were seeded in a 96-well tissue culture plate at 1×104 cell per well in 200 μL of 10% fetal bovine serum-DMEM. Cells achieving 70%–80% confluence were exposed to polymer at different doses for 24 hours followed by addition of 20 μL of Cell Titer 96® Aqueous One Solution (Promega Corporation, Madison, WI, USA). After further incubation for 4 hours, the absorbance was measured at 490 nm using a Tecan Infinite 500 plate reader (Tecan Systems Inc, San Jose, CA, USA) to obtain the metabolic activity of the cell. Untreated cells were taken as controls with 100% viability and wells without cells as blanks. The relative cell viability was calculated by:
(Atreated − Abackground) ×100/(Acontrol − Abackground)
All viability assays were carried out in triplicate.
In vitro transfection
C2C12E50 and C2C12E23 myoblast cell lines expressing green fluorescent protein (GFP) were used for in vitro experimentation. Expression of GFP was controlled by effective skipping of the inserted human dystrophin exon 50 sequence (hDysE50) or mouse dystrophin exon 23 sequence (mDysE23).18 The C2C12E50 cell line was maintained in 10% fetal bovine serum-DMEM in a humidified 10% CO2 incubator at 37°C. Approximately 5×104 C2C12E50 cells per well in 500 μL of 10% fetal bovine serum-DMEM medium were seeded and allowed to grow until confluence of 70%. The cell culture medium was replaced before addition of a polymer/PMOE50 (fixed at 5 μg) formulation with varying ratios. Polyethylenimine (PEI) 25,000 and Endoporter were used as a comparison. Transfection efficiencies indicated by GFP production were recorded after 48 hours of incubation with an Olympus IX71 fluorescent microscope (Olympus America Inc, Melville, NY, USA) and digital images taken with the DP Controller and DP Manager software (Olympus America Inc). Transfection efficiency was also examined quantitatively using flow cytometry. Cells were washed twice with phosphate-buffered saline (PBS; 1×, pH 7.4). After removal of PBS, 0.2 mL of 0.05% trypsin-ethylenediaminetetraacetic acid was added, and the cells were incubated for 3 minutes at 37°C. Next, 1 mL of the growth medium was added, and cells were collected by centrifugation and resuspended in 0.5 mL of ice-cold PBS (1×, pH 7.4). Samples were run on a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). At least 1×104 cells were counted and analyzed with CellQuest Pro software package (BD Biosciences).
RT-PCR analysis for cell culture
Collected cells were initially washed twice with PBS, and RNA was extracted with TRIzol reagent (Invitrogen Corp) according to the manufacturer's instructions. RNA was stored at −80°C for later use. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using RT-PCR Master Mix (2X) (USB Corp, Cleveland, OH, USA) to amplify the sequence of interest. Next, 100 ng of template RNA was used for each 25 μL RT-PCR reaction. The primer sequences for the RT-PCR were eGFP5′, 5′-CAGAATTCTGCCAATTGCTGAG-3′ and eGFP3′, 5′-TTCTTCAGCTTGTGTCATCC-3′. The cycle conditions for reverse transcription were 43°C for 15 minutes and 94°C for 2 minutes. The reaction was then cycled 30 times at 94°C for 30 seconds, 65°C for 30 seconds, and 68°C for 1 minute. The products were examined by electrophoresis on 1.5% agarose gel.
Cellular uptake and intracellular localization
To study cellular uptake and intracellular localization, fluorescein-labeled PMO (GeneTools) was combined with polymers at predetermined ratios, followed by imaging under confocal microscopy. C2C12 cells were seeded onto eight-well glass Nunc Lab-Tek II chamber slides (Thermo Scientific, Waltham, MA, USA) at 5×103 cells/well, and cultured to 70% confluence before addition of polymer/PMO formulation for testing. Approximately 24 hours after addition of the samples, the cells were washed with warm PBS (1×, pH 7.4) to remove any residual polymer/PMO polyplex from cells and incubated with medium. Cells were also counterstained with Hoechst 33258 (Life Technologies, Grand Island, NY, USA) to label cellular nuclei. Cellular uptake was examined on a Zeiss LSM-710 inverted confocal microscope (Carl Zeiss Microscopy LLC, Thornwood, NY, USA), and the resulting images were analyzed for uptake and localization by single-channel images. Co-localization of polymer/PMO to the lysosome was visualized by merged channel images to assess the signal intensities of the labeled PMO and lysosomes relative to counterstained nuclei.
The polymer/PMO polyplex solution containing 5 μg of PMO was prepared at a weight ratio of 5/5 (PE/PMO) in 200 μL of medium and analyzed using a Philips CM-10 transmission electron microscope (Philips Electronic North America Corp, Andover, MA, USA). The corresponding polymer and PMO only were used as comparison. The samples were prepared using negative staining with 1% phosphotungstic acid. Briefly, one drop of sample solution was placed on a formvar and carbon-coated carbon grid (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 hour, and blotted dry, followed by staining for 3 minutes. Samples were analyzed at 60 kV. Digital images were captured with a digital camera system from 4 pi Analysis (Durham, NC, USA).
In vivo delivery and RT-PCR
This study was carried out in strict accordance with the recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols were approved by the Institutional Animal Care and Use Committee, Carolinas Medical Center (breeding protocol 10-13-07A; experimental protocol 10-13-08A). All injections were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.3,5,6,18
Animals and intramuscular injections
Dystrophic mdx mice aged 4–5 weeks were used for in vivo testing (five mice each in the test and control groups) unless otherwise stated. The PMOE23 (5′-GGCCAAACCTCGGCTTACCTGAAAT-3′) targeting the boundary sequences of exon and intron 23 of the mouse dystrophin gene (GeneTools) was used. For intramuscular injections, 2 μg of PMOE23 with or without polymer was used in 40 μL of saline for each tibialis anterior muscle. The muscles were examined 2 weeks later, then snap-frozen in liquid nitrogen-cooled isopentane and stored at −80°C.
Total RNA was extracted from the muscle after dissection, and 100 ng of RNA template was used for a 50 μL RT-PCR with the RT-PCR Master Mix (2X) system (USB Corp, Cleveland, Ohio, USA). The primer sequences for the RT-PCR were Ex20Fo 5′-CAGAATTCTGCCAATTGCTGAG-3′ and Ex26Ro 5′-TTCTTCAGCTTGTGTCATCC-3′ for amplification of mRNA from exons 20 to 26. The conditions were 43°C for 15 minutes, 94°C for 2 minutes, then cycling 30 times at 94°C for 30 seconds, 56°C for 30 seconds, and 68°C for 1 minute. The products were examined by electrophoresis on 2% agarose gel. Bands with the expected size for the transcript with exon 23 deleted were extracted and sequenced. The intensity of the bands of the PCR-amplified products obtained from the treated mdx mouse muscles was measured using National Institutes of Health ImageJ software 1.42 and the percentage of exon-skipping was calculated with the intensity of the two bands representing both unskipped and skipped exons as 100%.
Antibodies, immunohistochemistry, and Western blots
Sections of 6 μm were cut from the muscles and stained with rabbit polyclonal antibody P7 for the dystrophin protein and detected by goat anti-rabbit immunoglobulins Alexa 594 (Invitrogen Corp). The maximum number of dystrophin-positive fibers in one section was counted using a BX51 fluorescent microscope (Olympus America Inc). Digital images were taken with the Olympus DP Controller and DP Manager software (Olympus America Inc) and the muscle fibers were defined as dystrophin-positive when more than two-thirds of the membrane of a single fiber showed continuous staining. Protein extraction and Western blot were performed as described previously.3,10,18 Briefly, the membrane was probed with NCL-DYS1 monoclonal antibody against dystrophin rod domain (1:200 dilution, Vector Laboratories, Burlingame, CA, USA) followed by horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (1:3,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the ECL™ Western blotting analysis system (Perkin-Elmer, Waltham, MA, USA). The intensity of the bands with appropriate size was measured and compared with that from normal muscles of C57BL mice using ImageJ software. Loading control of α-actin was detected by rabbit anti-actin antibody (Sigma Aldrich).
The data were analyzed for statistical significance using both one-way analysis of variance and the Student's t-test, with a value of P≤0.05 being considered statistically significant. All data are reported as the mean ± standard deviation.
The PEs applied in this study are commercially available and have been widely applied in the field of environment and biotechnology. Their structures and code names were shown in Figure 1.
Figure 1 Chemical structures, names, and codes for polyelectrolytes.
Abbreviation: MW, molecular weight.
PMO delivery in C2C12 myoblast cell lines expressing GFP/hDysE50
The C2C12E50 cell line was used to evaluate the efficacy of PEs for the delivery of PMO.18,28 This cell line expresses a GFP reporter, but its expression is disrupted by the insertion of the hDysE50. The expression of GFP in the reporter cells relies on the targeted removal of exon 50 by antisense oligonucleotides. First, we examined the cytotoxicity of the PEs using an MTS-based assay as shown in Figure 2. The toxicity of the PDDAC series including PE-1, PE-2, PE-3, and PE-4 was clearly size-dependent, being higher with increasing molecular weight of PDDAC. PE-5 (PADAC) and PE-6 (PBEBP) are structurally different from PDDAC. PE-5 contains insertion of around 55% polyacrylamide in composition; while PE-6 has a dispersed charged distribution due to the propyl urea spacer as compared with the PDDAC series. Protamine sulfate (PE-7) is a biocompatible and arginine-rich cell-penetrating polypeptide composed of only 50–110 amino acids, thus has a lower molecular weight and less toxicity than the PDDAC series.29–31
Figure 2 Viability of C2C12E50 cells after treatment with PEs at three doses (4, 10, 20 μg/mL from left to right for each polymer; PEI 25,000 was used as the comparison) determined by MTS assay. The cells were seeded in 96-well plates at an initial density of 1×104 cells/well in 0.2 mL of growth medium. The results are presented as the mean ± standard deviation (n=3, Student's t-test, *P≤0.05 compared with untreated cells).
Abbreviations: PEs, polyelectrolytes; PEI, polyethylenimine; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium].
However, all PEs showed much lower cytotoxicity against PEI 25,000 at the same dose used. Viability dropped to less than 25%, 38%, and 62% for the cells treated with PEI 25,000 at a concentration of 20, 10, and 4 μg/mL, respectively. In contrast, all PEs except PE-4 at a dose of 20 μg/mL showed cell viability over 75%. PE-4 had the highest toxicity; however, over 67% of cells remained alive after treatment at a dose of 20 μg/mL. The relatively higher toxicity of PE-4 is probably related to its higher molecular weight and higher positive charges when compared with other PEs.
We next examined the effect of the PE polymers on exon-skipping of PMO. The PMO sequence, PMOE50 (5′-AACTTCCTCTTTAACAGAAAAG CATAC-3′) with previously confirmed efficacy for targeted removal of human dystrophin exon 50 was used.18–20 C2C12E50 GFP reporter cells were treated with a fixed amount (5 μg) of PMOE50 in 500 μL of 10% fetal bovine serum-DMEM medium formulated with each polymer at four different doses (1, 2, 5, and 10 μg). Transfection efficiency was examined by fluorescence microscopy. The results showed that almost all PE polymers, even at 1 μg, improved GFP expression compared with the PMOE50 alone. The highest levels of GFP expression were achieved at a dose of 2–5 μg with most PEs, especially with the PDDAC series, reaching up to 90% with PE-2/3/4 at the dose of 2 μg and comparable to or higher than Endoporter-mediated delivery (5 μg, effective dose in vitro, commercial reagent produced by GeneTools). A dose-dependent GFP expression is also illustrated by PE-3 at the doses of 1, 2, 5 μg (Figure 3). In contrast, less than 5% of the cells were GFP-positive when treated with PMOE50 alone. The exon-skipping efficiency remained higher at the 10 μg dose of PEs, but some toxicity was observed with PE-3/4. The transfection efficiency and cell viability of the PE/PMO formulation at the PE doses (1, 2, 5 μg) mixed with PMO (5 μg) in 500 μL of medium were also quantified by flow cytometry with Endoporter (5 μg) and PEI 25,000 (2 μg) as controls (Figures 3 and 4). GFP expression resulting from PDDAC-mediated PMO delivery was noted to be up to 20-fold higher when compared with PMO alone. The PDDAC series (PE-1/2/3/4) has more positive charges compared with PE-5 or PE-6 at the same weight dose. PE-5 has around 55% polyacrylamide in the main chain compared with the PDDAC series, thus certainly contains a reduced surface charge when forming a PE/PMO polyplex; PE-6 has a dispersed charged distribution as compared with the PDDAC series. The low transfection efficiency with protamine sulfate is likely due to a much lower molecular size, thus limited charge groups within the polyplex.
Figure 3 Delivery efficiency and toxicity of PMOE50/PE complexes in a C2C12E50 cell line determined by fluorescence microscopy and fluorescence-activated cell sorting analysis.
Notes: (A) Representative fluorescence images of PMO-induced exon-skipping in the C2C12E50 cell line. The images were taken 48 hours after treatment. Original magnification, 100×. (B) Transfection efficiency of PMO formulated with PEs (one-way analysis of variance test, #P≤0.05 indicates a significant difference between PE groups; Student's t-test, *P≤0.05 compared with PMO only). (C) Cell viability (one-way analysis of variance test, P=0.585, no significant difference between PE groups was found; Student's t-test, *P≤0.05 compared with untreated cells). In this test, 5 μg of PMOE50 were formulated with PEs (1, 2, 5 μg), and PEI 25,000 (2 μg), and Endoporter (5 μg) was formulated as the control in 0.5 mL of 10% fetal bovine serum-Dulbecco's Modified Eagle's Medium, respectively. The results are presented as the mean ± standard deviation, in triplicate.
Abbreviations: PEs, polyelectrolytes; PEI, polyethylenimine; PMO, phosphorodiamidate morpholino oligomer.
Figure 4 Flow cytometry diagram. The upper panel shows the flow cytometry dot plots (FSC-SSC) and the lower panel shows the histograms (FL).
Abbreviations: PE, polyelectrolyte; PEI, polyethylenimine; PMO, phosphorodiamidate morpholino oligomer; FSC, forward scatter; SSC, side scatter; FL, flourescence.
In order to assess the delivery potential of the PEs for PMO exon-skipping in muscle fibers, rather than in myoblasts, the PEs were also tested in the mouse dystrophin exon 23 reporter C2C12 myoblast cell (C2C12E23). GFP reporter expression is driven by a muscle creatine kinase promoter, therefore allowed us to test the potential for PMO delivery in differentiating or differentiated myotubes.18 Cells reaching around 70% confluence were incubated in the differentiation medium for 2 days and then treated with PE-formulated PMOE23. The results showed a similar trend as that obtained in C2C12E50 cells, with PE-2, PE-3, and PE-4 achieving higher GFP expression than other PEs, as illustrated in Figure 5 by fluorescence images and RT-PCR detection of exon 23 skipping. The levels of exon 23 skipping were 56.4%, 64.1%, 59.0%, 32.6%, 24.7%, 38.5%, 1.8%, 23.7%, and 10.1% for PE-1, PE-2, PE-3, PE-4, PE-5, PE-6, PE-7, Endoporter-formulated PMO, and PMO only, respectively. The results indicate the importance of molecular size, chemical structure, and positive charge distribution in the vector microstructure for both delivery efficiency and toxicity.
Figure 5 Green fluorescent protein expression induced by PMOE23 (5 μg) formulated with PEs in C2C12E23 cells (PE or Endoporter 5 μg and PMOE23 5 μg in 0.5 mL of 10% fetal bovine serum-Dulbecco's Modified Eagle's Medium after 48 hours treatment). Upper panel shows fluorescence detection of green fluorescent protein expression, original magnification, 100×; Lower panel shows reverse transcription-polymerase chain reaction of exon 23 skipping.
Abbreviations: PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.
To examine whether PEs improve the cell uptake of PMO, we examined the intracellular localization of the PE/PMO polyplex. PE was complexed with 3′-carboxyfluorescein-labeled PMO at a weight ratio of 5/2. The presence of PE appeared to affect the pathway of PMO uptake, as demonstrated by confocal microscopy analysis. PMO alone distributed evenly within the cytoplasm of the cells, in agreement with a reported passive diffusion model.16 Signals for PMO were considerably stronger in cells treated with PE-4 and visualized as punctuates within the cytosol and especially around the nucleus (Figure 6). These results suggest that the presence of PEs alters the route of PMO internalization, probably eliciting endocytosis through formation of a complex.
Figure 6 Confocal microscopic images of C2C12 cells treated with FITC-PMO (2 μg) without and with PE-4 (5 μg). Nuclear staining with Hoechst 33258. The images were obtained under a magnification of 63×. Labeled PMO can be observed at lower levels throughout the treated cells; however, with the addition of PE-4, greater levels of labeled PMO can be observed with greater concentrations in the perinuclear space.
Abbreviations: PE, polyelectrolyte; FITC, fluorescein isothiocyanate; PMO, phosphorodiamidate morpholino oligomer.
Interaction between PE and PMO
The affinity between a polymer and an oligonucleotide is an important parameter for their efficient delivery into cells. Here, we chose the most effective PE-4 from the PDDAC series and PE-5 for PE/PMO polyplex examination under transmission electron microscopy. As shown in Figure 7, the PE-4 polymer alone formed particles of different sizes, likely because of aggregation, whereas the PMO oligonucleotides alone formed particles with a size below 50 nm, likely resulting from hydrophobic interactions and hydrogen-bond among the PMO molecules. At a weight ratio of 5/5, the PE-4/PMO polyplex formed spherical particles with an average diameter around 20–40 nm. The PE-5/PMO polyplex formed larger particles than did the PE-4/PMO polyplex. This is probably the result of aggregation related to the uncharged hydrophobic polyacrylamide fragments within PE-5. The mechanisms of interaction between PMO and the PE molecules are not clear, but the chemical nature of PMO likely creates a hydrophobic interaction with the PEs, and a possible hydrogen-bond interaction between them. The positively charged groups within the PEs are unlikely to play a key role in the interaction with PMO; nonetheless, the surface charges of the polyplex may stabilize it in a biological environment for a longer period than PMO alone.
Figure 7 Negatively stained transmission electron micrographs of PE (5 μg) with and without PMO (5 μg) complexes and PMO only (scale bar, 100 nm).
Delivery of PMO with PEs in vivo
We next evaluated the effect of the PE polymers on PMO delivery in vivo by intramuscular injection. PMOE23 targeting mouse dystrophin exon 23 was injected into each tibialis anterior muscle of mdx mice aged 4–5 weeks. The mouse contains a nonsense mutation in exon 23, preventing production of the functional dystrophin protein. Targeted removal of the mutated exon 23 is able to restore the reading frame of dystrophin transcripts, and thus the expression of the dystrophin protein. Based on the delivery performance of PEs in vitro, we chose 2 μg as an effective and safe dose, premixed with 2 μg of PMOE23 in 40 μL of saline. The treated tibialis anterior muscles were harvested 2 weeks later.
Immunohistochemistry showed that the PMOE23 alone induced up to 12% maximum dystrophin-positive fibers in one cross-section of the tibialis anterior muscle. The number of dystrophin-positive fibers increased dramatically in the muscles treated with PMOE23 mediated by PEs. The PDDAC series enhanced PMO-mediated exon-skipping with increasing molecular size. PE-3 and PE-4 achieved over 40% and 50% positive fibers respectively, ie, over fourfold as compared with PMO alone at the tested dose. Meanwhile, PE-5, PE-6, and PE-7 did not dramatically change the number of dystrophin-positive fibers (Figure 8). These results correlate well with the data in muscle cell lines in vitro, suggesting that the smaller PE molecule was less able to form an optimal complex with PMO, resulting in low transfection efficiency.32,33 PE-3 or PE-4 with higher transfection efficiency is probably due to larger molecular size, thus creating higher affinity binding sites with PMO. The positively charged PE-PMO complex is likely to be more stable in biological systems as the complex is expected to interact with various cells and biomacromolecules in the tissue and circulation, maintaining a longer circulation time.34,35 The levels of exon-skipping and corresponding dystrophin expression were also quantitatively determined by RT-PCR and Western blot, respectively. PMO formulated with PE-1, PE-2, PE-3, PE-4, PE-5, PE-6, PE-7 and PMO only achieved levels of exon-skipping at 4.1%, 30.5%, 22.9%, 25.5%, 0.2%, 20.8%, 0.8%, and 2.7%, respectively. Dystrophin protein expression levels were found to be 3.5%, 23.7%, 6.2%, 51.2%, 9.5%, and 3.7% of normal levels (taking muscles from C57 mouse as 100%) for PMO formulated with PE-1, PE-2, PE-3, PE-4, PE-6 and PMO only, respectively. Both quantitative and qualitative data therefore demonstrated the best performance with the PDDAC series, especially with PE-4. The results indicate the importance of molecular size and composition for the efficacy of delivery of uncharged PMO. However, more effective PEs are associated with higher toxicity, further highlighting the difficulty and complexity of developing non-viral oligonucleotide delivery agents for clinical use.
Figure 8 Restoration of dystrophin in tibialis anterior muscles of mdx mice (aged 4–5 weeks) 2 weeks after intramuscular injection.
Notes: (A) Dystrophin was detected by immunohistochemistry with rabbit polyclonal antibody P7 against dystrophin. Blue nuclear staining with 4,6-diamidino-2-phenylindole. Muscles treated with PMOE23 (2 μg) only was used as controls. All other samples were from muscles treated with 2 μg polymer and 2 μg PMOE23 in 40 μL saline. Original magnification, 100×. (B) The percentage of dystrophin-positive fibers in muscles treated with 2 μg PMOE23 with and without polymers (2 μg). The maximum numbers of dystrophin-positive fibers were counted in a single cross-section (n=5, one-way analysis of variance test, #P≤0.05, there were significant difference between PE groups; Student's t-test, *P≤0.05 compared with 2 μg PMO). (C) Detection of exon 23 skipping by reverse transcription-polymerase chain reaction. Total RNA of 100 ng from each sample was used for amplification of dystrophin mRNA from exon 20 to exon 26. The upper bands (indicated by E22 + E23 + E24) correspond to the normal mRNA, and the lower bands (indicated by E22 + E24) correspond to the mRNA with exon E23 skipped. (D) Western blots demonstrate the expression of dystrophin protein. Dystrophin detected with monoclonal antibody Dys 1. A loading control (α-actin) was used.
Abbreviations: Dys, dystrophin; PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.
In summary, the cationic PEs have been evaluated for the first time as vectors for antisense PMO delivery in vitro and in dystrophic mdx mice. The results show that the PDDAC series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, that is comparable to Endoporter-mediated PMO delivery in vitro. Significant enhancement of PMO delivery was also demonstrated in vivo, and up to fourfold with PE-4, when compared with PMO alone. No obvious local toxicity was observed at the test dose of PE-4. These data suggest that optimization of the molecular size, components, and density of the positive charge of PEs can achieve enhanced exon-skipping with PMO. PEs are therefore potential vehicles for antisense oligonucleotide delivery to achieve a therapeutic effect in diseases such as Duchenne muscular dystrophy.
The authors would like to thank Drs David M Foureau and Fei Guo for their technical assistance with the flow cytometry and analysis, and Mrs Daisy M Ridings and Ben Wagner from the Electron Microscopy Core Laboratory for the negative staining and transmission electron micrographs. The authors also gratefully acknowledge the financial support provided by the Carolinas Muscular Dystrophy Research Endowment at the Carolinas HealthCare Foundation and Carolinas Medical Center, Charlotte, NC, USA.
The authors declare no conflicts of interest in this work.
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Mendell JR, Rodino-Klapac LR, Sahenk Z, et al. Eteplirsen for the treatment of Duchenne muscular dystrophy. Ann Neurol. 2013;74:637–647.
Cirak S, Arechavala-Gomeza V, Guglieri M, et al. Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet. 2011;378:595–605.
Fletcher S, Honeyman K, Fall AM, Harding PL, Johnsen RD, Wilton SD. Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide. J Gene Med. 2006;8:207–216.
Malerba A, Sharp PS, Graham IR, et al. Chronic systemic therapy with low-dose morpholino oligomers ameliorates the pathology and normalizes locomotor behavior in mdx mice. Mol Ther. 2011;19:345–354.
Summerton J, Weller D. Morpholino antisense oligomers design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 1997;7:187–195.
Yano J, Smyth GE. New antisense strategies: chemical synthesis of RNA oligomers. Adv Polym Sci. 2012;249:1–48.
Hu Y, Wu B, Zillmer A, et al. Guanine analogues enhance antisense oligonucleotide-induced exon skipping in dystrophin gene in vitro and in vivo. Mol Ther. 2010;18:812–818.
Wang MX, Wu B, Tucker JD, Lu P, Cloer C, Lu QL. Evaluation of Tris[2-(acryloyloxy)ethyl]isocyanurate cross-linked polyethylenimine as antisense morpholino oligomer delivery vehicle in cell culture and dystrophic mdx Mice. Hum Gene Ther. 2014;25:419–427.
Wang MX, Wu B, Lu P, Cloer C, Tucker JD, Lu QL. Polyethylenimine modified pluronics (PCMs) improve morpholino oligomer delivery in cell culture and dystrophic mdx mice. Mol Ther. 2013;21:210–216.
Liverpool TB, Stapper M. The scaling behaviour of screened polyelectrolytes. Europhys Lett. 1997;40:485–490.
Dobrynin AV, Colby RH, Rubinstein M. Scaling theory of polyelectrolyte solutions. Macromolecules. 1995;28:1859–1871.
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Dautzenberg H. Polyelectrolyte complex formation: Role of a double hydrophilic polymer. Macromol Chem Phys. 2000;201:1765–1773.
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Sorgi FL, Bhattacharya S, Huang L. Protamine sulfate enhances lipid-mediated gene transfer. Gene Ther. 1997;4:961–968.
Sazani P, Kang SH, Maier MA, et al. Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucleic Acids Res. 2001;29:3965–3974.
Balhorn R. The protamine family of sperm nuclear proteins. Genome Biol. 2007;8:227.
Laufer SD, Restle T. Peptide-mediated cellular delivery of oligonucleotide-based therapeutics in vitro: quantitative evaluation of overall efficacy employing easy to handle reporter systems. Curr Pharm Des. 2008;14:3637–3655.
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Tricyclea semicinerea är en tvåvingeart som beskrevs av Mario Bezzi 1908. Tricyclea semicinerea ingår i släktet Tricyclea och familjen spyflugor. Inga underarter finns listade i Catalogue of Life.

Källor

Spyflugor
semicinerea

redpajama_set_name: RedPajamaWikipedia


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Columnist: Trump's success with Black voters could spell trouble for Joe Biden
Democrats have historically enjoyed wide margins of support from Black voters — but President Donald Trump seems to be turning the tide in a way Joe Biden and company never expected.
According to the Washington Examiner's Paul Bedard, polls have shown "consistent" support for President Trump among Black voters "at levels higher than those of previous Republican candidates" — and that could spell trouble for Biden as he seeks to unseat the incumbent president this fall.
Biden falls short
Based on the results of previous elections, Democrats who hope to win the White House should receive at least 90% of votes from the Black community, according to Bedard. The columnist reported:
In 2008 and 2012, Barack Obama won 96% and 93% of the [B]lack vote. Hillary Clinton's 89% to Trump's 9% victory among [B]lacks was not enough to win the Presidency.
As things stand, Biden is more on track with Clinton than his former running mate. According to a poll conducted in early September by Newsmax/John Zogby Strategies, Trump currently has the support of about 14% of likely voters who are Black.
Biden, by contrast, has drawn about 77% of that demographic's support, well short of the roughly 90% most analysts predict is necessary to assure victory, Bedard noted, citing pollster John Zogby.
"The numbers from likely [B]lack voters are nowhere near where they should be," Zogby wrote of Biden in his Main St.-K St. IntelligenSEER. "This alone could potentially cost Biden the election."
Casting a shadow
Trump has also seen success particularly among Black voters in the 18-to-24 age demographic: though many would expect Biden to have that category all to himself, Trump has garnered the support of 38% of voters polled in that group, and Biden holds just 61% support.
What's more, according to Zogby, Democrats who won in prior presidential elections also often saw the overwhelming support — 60% or more — of women and young voters of all races, but there seems to be a decided lack of enthusiasm for Biden. He bests Trump just 49% to 38% among women, and holds the support of 50% of young voters to Trump's 31%.
"If Biden's support from [B]lacks remains this low, that alone could cost the election. Then compile his deficit with the youth vote and women, and this casts a shadow over Biden," Zogby wrote in his recent report. "He is leading by three points today, but remember Clinton won the popular vote by two points in 2016 and still lost."
As for Trump, Zogby surmised that his law-and-order message and promises to keeping local police departments intact could work in his benefit by continuing to draw support away from Biden among the voters most impacted by recent protests.
"As violence continues, as cities go up in flames, and as crime continues to soar in large cities, the tables are likely to turn on Biden as more youth, women, and [B]lacks either choose not to turn out or just enough cast their vote for Trump," Zogby wrote. What's more, the pollster reported that while Biden retains a slim lead when survey respondents are asked who they support, the numbers change when respondents are asked: Who do you think will win?
"Overall, 43% say they believe Trump will win, and 40% say they believe Biden will win," Zogby wrote, concluding:
For some, this could be fear; for others, it is a reflection of the widely held belief that Trump is a winner and performs well under pressure. But it further bolsters the point made earlier that as of today, Biden's popularity is on the wane and that he is desperate need of a major boost.
Ben Marquis
Ben Marquis is a staff writer for Conservative Institute.

redpajama_set_name: RedPajamaCommonCrawl


The City of Calgary Composting Facility (CoCCF) has, to date, been one of the most interesting projects I've worked on in my career. This isn't just because it's the first LEED® v4 project that I've coordinated, although that was a big part. I love working on projects that positively impact our communities.
But before we get into the project, let's talk about LEED® v4. Yes, there's a new version of the LEED® rating system. How is it different? One of LEED® v4's biggest differentiators is its focus on integration. This really came into play in the CoCCF project, as it strayed from typical project delivery methods, and engaged all members of the project team—including the contractor and operator—at the onset, and has continued this collaboration throughout the project life. Bottom line, everyone is committed to a responsible design from the get go, and supports it right through to completion.
So what does this look like in practice? The City of Calgary selected the LEED® v4 rating system, targeting Gold certification for a portion of the CoCCF, in support of the City's Sustainable Building Policy and their commitment to divert 80% of waste from landfills by 2020. At over 800 square metres, the CoCCF's Administration and Education (A&E) Annex, presented a great opportunity for the City to implement more stringent (compared to earlier versions of LEED®) water and energy targets. Our design team was brought on board to tackle the aspirational standards that the integrative design processes required to achieve LEED® v4 certification. We were determined to go for Gold!
For the CoCCF—a Design-Build-Operate project—it meant a diverse team of Stantec architectural, mechanical, electrical, structural, civil, sustainability, and geotechnical engineering experts needed to collaborate from the beginning with the contractors and the building operators.
Pursuing LEED® v4 Gold allowed us to maximize interrelationships between architectural, electrical, mechanical, and structural components and exterior environmental systems to create a high performance, cost effective building. With the entire team involved from the beginning, communication was at the forefront, making key decisions easy and efficient.
Are there still challenges? Of course! But another benefit of structuring a project around LEED® v4 is you have the combined creativity of the entire design team to find solutions. For example, water is a big part of the composting process. We wanted to avoid relying on potable water to make this process work because why waste perfectly good water that falls from the sky? Our team incorporated the facility's design into the surrounding property to make sure 100% of rainwater landing on the site flows into a stormwater pond that and directed into the facility to assist with composting. And, why stop there? Our team saw an opportunity to use a portion of the rainwater to flush toilets in the A&E Annex to further reduce reliance on the potable water supply. That's the kind of collaboration that gets you to LEED® v4.
One of my co-workers commented that one of the best feelings is going into a building and knowing that you were part of the team that brought this facility all the way from concept to reality. I can certainly relate because working on a project like the CoCCF and using the LEED® v4 system takes that feeling of pride up a notch for me. This is a project that affects all of us in Calgary. I'm so glad that I've been able to work with a diverse team on a project dedicated to making our community process waste and use water more responsibly.
Erica Baranik is a Sustainability Analyst in Calgary's Buildings team and is passionate about how building design impacts the environment. She spoke about her experience working on the City of Calgary Composting facility at the Canada Green Building Council conference.


Superdry men's Lucky Aces t-shirt. This crew neck t-shirt features a large puff print Superdry Lucky Aces logo design on the chest. There is also a cracked finish number print on the sleeve, a logo patch above the hem and the t-shirt is finished with a Superdry logo tab on the sleeve.


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Tag Archives: jewish
5 Comments Posted in history, holidays, politics, Uncategorized Tagged Chanukah, civil war middle east, jewish, miracles
Missing Paul Wellstone
Yesterday I attended Candidate Training with Kevin Winchell in Palm Beach County. He referred to Wellstone Training and it for a moment I was terrified that I had not thought about Paul Wellstone for a very long time. TIme passed and people become a blur because they are not in front of us on 24 hour news cycle.
This is one person we can never ever forget.
http://en.wikipedia.org/wiki/Paul_Wellstone
Paul David Wellstone (July 21, 1944 – October 25, 2002) was an American academic and politician who represented Minnesota in the United States Senate from 1991 until his death in a plane crash in 2002. A member of the Democratic Farmer-Labor Party, Wellstone was a leading spokesman for the progressive wing of the national Democratic Party.
In 1998, Wellstone formed an exploratory committee and a leadership PAC, the Progressive Politics Network, that paid for his travels to Iowa and New Hampshire, two early primary states in the nomination process. He spoke before organized labor and local Democrats, using the slogan, "I represent the democratic wing of the Democratic Party". Governor Howard Dean later incorporated that phrase into his stump speech in the 2004 US presidential election.[3]
On January 9, 1999, Wellstone called a press conference at the Minnesota State Capitol. Despite expectations that he would announce his candidacy, he stated he could not muster the stamina necessary for a national campaign, citing chronic back problems he ascribed to an old wrestling injury. His pain was later diagnosed as multiple sclerosis.
On October 25, 2002, Wellstone, along with seven others, died in an airplane crash in northern Minnesota, at 10:22 a.m. He was 58 years old. The other victims were his wife, Sheila; one of his three children, Marcia; the two pilots, his driver, and two campaign staffers. The airplane was en route to Eveleth, where Wellstone was to attend the funeral of Martin Rukavina, a steelworker whose son Tom Rukavina served in the Minnesota House of Representatives. Wellstone decided to go to the funeral instead of a rally and fundraiser in Minneapolis attended by Mondale and fellow Senator Ted Kennedy. He was to debate Norm Coleman in Duluth, Minnesota, that same night.
In 2007, former First Lady Rosalynn Carter joined with David Wellstone to push Congress to pass legislation regarding mental health insurance.[54] Wellstone and Carter worked to pass the "Paul Wellstone and Pete Domenici Mental Health Parity and Addiction Equity Act of 2008" which requires equal coverage of mental and physical illnesses when policies include both types of coverage; both testified before a House subcommittee regarding the bill in July 2007.[54] David said of his father, "Although he was passionate on many issues, there was not another issue that surpassed this in terms of his passion."[54] Because Paul Wellstone's brother had suffered from mental illness, Wellstone had fought for changes in mental health and insurance laws when he reached the Senate.[54]
http://en.wikipedia.org/wiki/Public_Law_110-343#Paul_Wellstone_and_Pete_Domenici_Mental_Health_Parity_and_Addiction_Equity_Act_of_2008
The Paul Wellstone and Pete Domenici Mental Health Parity and Addiction Equity Act of 2008 (a part of Division C) mandates that if U.S. health insurance companies provide coverage for mental health and substance abuse, the coverage must be equal for conditions such as psychological disorders, alcoholism, and drug addiction.[4][24][25] This act continues and expands upon the previous Mental Health Parity Act of 1996. It states that financial requirements such as deductibles and copayments and lifetime or dollar limits to mental health benefits and substance abuse disorder benefits should be no more restrictive than those on medical and surgical benefits.[26] MHPAEA applies to employer-sponsored health insurance plans with more than 50 employees, though parity is also extended to small group and individual plans under the Affordable Care Act.
He would have loved Obama Care because people like his brother could get the care he deserved.
http://www.theatlantic.com/politics/archive/2012/10/paul-wellstones-legacy-10-years-later/264086/
Al Franken wrote
"When today's progressives remember Paul, they tend to focus on his sheer political courage. Paul was the only senator up for re-election in 2002 to vote against the Iraq War — a vote he told people he was convinced would cost him his job.
One of Paul's most famous quotes is this: "Politics is not about power. Politics is not about money. Politics is not about winning for the sake of winning. Politics is about the improvement of people's lives.
The big fights — war and peace, justice and liberty — are important. But there aren't any small fights. And where Paul made the biggest impact — where his work resulted in the greatest improvement of people's lives — was on issues that don't usually lead anyone's stump speech: mental health, domestic violence, homelessness among veterans.
Paul's brother, Stephen, had struggled with mental illness since he was 19. And Paul knew that many people who suffered from mental health problems were forced to suffer all over again because their insurance companies wouldn't cover the treatments they needed. Paul's own parents spent 20 years paying down the bill for Stephen's treatment.
So he found a Republican with a similar first-hand understanding of the problem, New Mexico Senator Pete Domenici, and the two introduced legislation that would require insurance companies to treat mental health the same way they did physical health. Paul didn't live to see mental health parity become law — but it did (thanks in large part to another Republican colleague, Minnesota's Jim Ramstad), and that crusade has improved, even saved, millions of lives.
hat's why Paul's political career began by organizing college students and low-income women to fight for better anti-poverty measures, and organizing farmers to fight against utility companies planning to install power lines on their land. And that's why the organization that bears his name and carries forward his legacy, Wellstone Action, isn't a think tank or a super PAC. Wellstone Action trains organizers — not just to articulate progressive values, but to do the "prosaic, practical work" that makes a difference."
First campaign "Looking for Rudy"
Opposing the Iraq War
"Fast Paul" awesome campaign ad
I'll never forget the day that Paul Wellstone died. I can never forget this.
and I went to order his book from Amazon
http://www.amazon.com/The-Conscience-Liberal-Reclaiming-Compassionate/dp/081664179X
Amazon reminded me that I bought this book before. I bought it October 2002.
Let's not forget this awesome man.
Leave a comment Posted in environmental, mental illness, politics Tagged idologue, iraq war, jewish, librael, New Mexico Senator Pete Domenici, paul wellstone, progressive, universal health care, wellstone training


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The Critical-Theory Guide to that Time Zizek Pissed Everyone Off (Again).
May 22, 2013 Sir Giggleton	2 Comments
Trollin
Zizek is one of the more controversial authors of our time. He seems to have no problem calling vegetarians "degenerates" or shoeing away most of his fans. So it's no surprise that he's gained a lot of fans and a lot of critics. One fan, a nice fellow named Santiago Zabala, published a nice long love letter to Zizek's philosophy entitled "Slavoj Zizek and the Role of the Philosopher." And, like the start of any good flame war, the article was summarily shit on by most post-colonial critics everywhere. Why? Well, Zizek is pretty damn Eurocentric.
If you've been confused by a recent spat between Zizek and, well, everyone, you're not alone. We previously spoke about Zizek responding to his critics, but without proper context, you may have been lost. We therefore present the Critical-Theory guide to that time Zizek pissed everyone off (again).
This war of words occurred mainly over Al-Jazeera's opinion blog, with the exception of Zizek's public lecture at the Birbeck Institute for the Humanities, because he's too cool for Al-Jazeera. Folks featured are Santiago Zabala, Hamad Dabashi, Slavoj Zizek, Walter Mignolo and Michael Marder.
Initial Salvo: Santiago Zabala
In: Slavoj Zizek and The Role of the Philosopher
Santiago Zabala begins this dispute by long ode to that man that is Slavoj Zizek. He describes Zizek as one "fuse together Martin Heidegger's "fundamental ontology", Francis Fukuyama's "end of history" and Naomi Klein's "shock doctrine" in order to undermine our liberal and tolerant democratic structures … a practice few intellectuals are capable of". His engagement with the specific struggles of Slovenia, Julian Assange and Palestine and the broader theoretical horizon of communism are what makes him, in Zabala's words, a "thinker of our age", able to delve into the mundane sequence of political and cultural events and artifacts and integrate it into the theoretically complex worlds of the theories of Marx, Lacan and Hegel.
The Reponse – Hamid Dabashi
In: Can Non-Europeans Think?
Hamid Dabashi takes with the uh, geographically specific, nature of Zabala's philosophical shout-outs. Zabala's piece reads:
There are many important and active philosophers today: Judith Butler in the United States, Simon Critchley in England, Victoria Camps in Spain, Jean-Luc Nancy in France, Chantal Mouffe in Belgium, Gianni Vattimo in Italy, Peter Sloterdijk in Germany and in Slovenia, Slavoj Zizek, not to mention others working in Brazil, Australia and China.
This, Dabashi argues, is a location of thought in the global north, and a degradation of the value of thought emanating from the global south. He argues that the fact that the European intellectuals receive the distinction of being named while the others are merely clumped into a large category portrays a larger bias in which these philosophers are declared ethnic and are discounted or sectioned off into an independent area of study, whereas the European's work is declared philosophy. Rather, he argues that philosophy such as Zizek's, Critchley's or Butler's should be offered as European philosophy, not the universal depiction of reality which he claims Zabala attempts to sell Zizek's approach as.
Then Zabala is Like "Communism is Alive! Long Live Communism!"
In: "Zizek and the Communist Horizon"
Zabala responds to Dabashi's contention that he is Eurocentric by arguing that Communism, as offered by Zizek, is a universal lens through which to interpret reality. Against the particularity, he argues that we can engage with the communist analytic method of dialectical materialism in everyday life and act through that lens. This is not through the application of stale maxims, but of the adoption of a certain approach or way of engaging with reality that lends coherence to the scattered sequences of events such as 9/11 or the Financial Meltdown of 2008. In response to the real conditions of the oppression by capitalism and the impending extinction due to capitalistic exploitation, we are called to resist this in a universalistic, communistic manner. Instead of acting to curing the symptoms of capitalisms domination in totalitarianism, instability and terror, we have to get to the root of the problem: capitalism.
And Then Walter Mignolo Chimes In
Lol, wut?
Walter Mignolo argues that the work of these de-colonial philosophers who are underrepresented in the original article are more relevant to the concerns of their people. He begins by arguing that Zabala believes in a hierarchy of work and struggles rather than a non-hierarchical relationship between struggles, which he believes is embodied by Dabashi. He then responds to Zizek's article "A Leftist Plea for 'Euro-centrism'", and specifically quotes Zizek's first line:
When one says Eurocentrism, every self-respecting postmodern leftist intellectual has as violent a reaction as Joseph Goebbels had to culture – to reach for a gun, hurling accusations of proto-fascist Eurocentrist cultural imperialism. However, is it possible to imagine a leftist appropriation of the European political legacy?
Rather, Mignolo responds:
"A self-respecting decolonial intellectual will reach instead to Frantz Fanon: 'Now, comrades, now is the time to decide to change sides. We must shake off the great mantle of night, which has enveloped us, and reach, for the light. The new day, which is dawning, must find us determined, enlightened and resolute. So, my brothers, how could we fail to understand that we have better things to do than follow that Europe's footstep.'"
To Mignolo, the relevance of Zizek's ideas is not universal but is rather so only to the European intellectuals from which these ideas originated. Rather each population requires its own solutions to its own problems, which can be a disengagement from capitalism or communism or both. He cites Bolivia where "communism" is a problematic social arrangement for the Aymaras and Quechuas of the area.
And Bam! Zizek Tells Mignolo "Fuck You"
In his public lecture at the Birbeck Institute of Humanities, Zizek responds to Mignolo's arguments.
He begins by disputing the ability to solve the problems of capitalism of the philosophers Mignolo elevates. He specifically takes issue with Wang Hui, who he characterizes as a "Left Friedman" who decenters the struggle against capitalism from capitalism itself, but instead to the evil monopolies which destroy proper free-market exchange. Zizek reads this as the apologia that thought not centered upon the rejection of capitalism makes for it.
He proceeds by contesting Mignolo's invocation of Fanon. Rather than a sanitized Fanon who simply opts for symbolic acts and is a true expression of an Algerian de-colonial agency, he emphasizes the violent, universalizing Fanon who utilizes the "Eurocentric" work of Lacan and Hegel. He uses an extended quote by Fanon to make the argument that when he faces "de-colonial" intellectuals like Mignolo, he reaches instead for Fanon:
I am a man and what I have to recapture is the whole past of the world, I am not responsible only for the slavery involved in Santo Domingo, every time man has contributed to the victory of the dignity of the spirit, every time a man has said no to an attempt to subjugate his fellows, I have felt solidarity with his act. In no way does my basic vocation have to be drawn from the past of peoples of color. In no way do I have to dedicate myself to reviving some black civilization unjustly ignored. I will not make myself the man of any past. My black skin is not a repository for specific values. Haven't I got better things to do on this earth than avenge the blacks of the 17th century?
I as a man of color do not have the right to hope that in the white man there will be a crystallization of guilt towards the past of my race. I as a man of color do not have the right of stamping down the pride of my former master. I have neither the right nor the duty to demand reparations for my subjugated ancestors. There is no black mission. There is no white burden. I do not want to be victim to the rules of a black world. Am I going to ask this white man to answer for the slave traders of the 17th century? Am I going to try by every means available to cause guilt to burgeon in their souls? I am not a slave to slavery that dehumanized my ancestors. It would be of enormous interest to discover a black literature or architecture from the 3rd century B.C, we would be overjoyed to learn of the existence of a correspondence between some black philosopher and Plato, but we can absolutely not see how this fact would change the lives of 8 year old kids working the cane fields of Martinique or Guadeloupe. I find myself in the world and I recognize I have one right alone: of demanding human behavior from the other.
What is rather the proper path of these struggles is to recognize the universalizing possibilities of communism and the truth of dialectical materialism, according to Zizek.
Parting Shot: Michael Marder
In: A Post-Colonial Comedy of Errors
Marder largely responds to the article from Dabashi by arguing that Dabashi's chutzpah sets him at the center of the world while ignoring that Europe itself is full of divisions and minorities such as Southern Europeans and Balkan states, where Zabala and Zizek hail from, respectively. He also argues that the general depiction of the list of authors as Eurocentric ignores the variations of each author and their anti-hegemonic orientation which demands a more individual analysis and respect to the work actually put out rather than location. As Anti-Eurocentrism has oriented itself away from these "grand narratives" so too have these thinkers. Rather, much as in Shakespeare's Comedy of Errors where Dromio is beaten for deeds not committed, these philosophers are accused of crimes not committed.
Santiago Zabala is a professor at the University of Barcelona. He's published books such as Hermeneutic Communism and the Remains of Being.
Walter Mignolo is a professor at Duke University whose work focuses on semiotics and literary theory. He has authored books such as The Darker Side of Western Modernity and The Darker Side of the Renaissance.
Slavoj Zizek is Slavoj Zizek. His books include The Year of Dreaming Dangerously and Living in the End Times.
Michael Marder is a professor at the University of the Basque Country, Vitoria-Gasteiz. His books include Plant-Thinking: A Philosophy of Vegetal Life and The Event of the Thing.
Hamid Dabashi is a professor at Columbie University. His books include The World of Persian Literary Humanism and The Arab Spring: The End of Postcolonialism
DabasihMignoloPostcolonialismSlavoj ZizekZabala
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No. 14 UCLA's late rally falls short in home finale, Bruins lose 38-33 to No. 19 Arizona St.
UCLA wide receiver Grayson Mazzone, left, injures his left knee as he runs the ball while Arizona State safety Alden Darby defends during the second half an NCAA college football game, Saturday, Nov. 23, 2013, in Pasadena,Calif. Mazzone needed to be helped off the field. (AP Photo/Mark J. Terrill)(The Associated Press)
Arizona State running back D.J. Foster, left, is tackled by UCLA safety Randall Goforth during the second half an NCAA college football game, Saturday, Nov. 23, 2013, in Pasadena, Calif. (AP Photo/Mark J. Terrill)(The Associated Press)
PASADENA, Calif. – UCLA stumbled off the field at halftime trailing by 22 points, its Pac-12 South title hopes all but buried by Taylor Kelly and Arizona State's prolific offense.
Although the Bruins managed to turn a potential blowout into a bit of a nail-biter late, they'll spend a long winter regretting the half that got away in their biggest game of the season.
Kelly passed for 225 yards and a touchdown and rushed for 99 yards and another score, and No. 19 Arizona State hung on in the fourth quarter to clinch the division title with a 38-33 victory over No. 14 UCLA on Saturday night.
Brett Hundley passed for 253 yards and two touchdowns for UCLA (8-3, 5-3), which had hoped to earn a third straight trip to the conference championship. But a 35-13 halftime deficit was too much to overcome, even in a stadium where the Bruins hadn't lost all season.
Another ugly bit of math: Ka'imi Fairbairn missed two makeable field goals in the Bruins' five-point loss.
"These guys, they just fought back," UCLA coach Jim Mora said. "We were in it. We had shots. Those are the what-ifs and the could-have-beens, and they don't matter. We just have to get better from it. And we will."
Marion Grice had 95 yards rushing and 72 yards receiving for the surging Sun Devils (9-2, 7-1 Pac-12), who will face Stanford in the league title game in two weeks.
The Bruins couldn't overcome their dismal start despite repeatedly getting outstanding field position from Ishmael Adams, who had 234 total yards in kick returns. Myles Jack rushed for 86 yards and a touchdown while the freshman linebacker played almost exclusively on offense.
Kelly and the Sun Devils' offense did nearly all of its damage before halftime. UCLA gave up just three points and 91 yards in the second half, but the Bruins couldn't score on their final two drives.
"Nothing we hadn't seen before," UCLA linebacker Anthony Barr said. "Just their tempo in the first couple of series hurt us, and their quarterback hurt us with his legs. We made those adjustments, but it was too little, too late."
If Arizona State beats Arizona in the Territorial Cup game next week, the Sun Devils will host Stanford — a possibility almost nobody anticipated just a couple of weeks ago.
"We got a chance to play for a trip to the Rose Bowl, which I've dreamed of all my life," Kelly said in the Rose Bowl tunnel. "We've got a couple more to go, and then we can be there."
Jaelen Strong had six catches for 91 yards and a score, but Arizona State blew most of its halftime lead over the Bruins. In the schools' first meeting as two ranked teams since 1986, Arizona State earned the right to play for its first Rose Bowl berth in 17 years.
"I've been doing this for 28 years, and that's the most impressive display of heart by a team that I've ever seen," said Arizona State coach Todd Graham, who acknowledged getting choked up on the field for maybe the second time in his adult life. "To watch these kids lay it on the line with that kind of heart was overwhelming to me."
UCLA trailed 38-27 entering the fourth quarter, but Shaq Evans caught a 27-yard TD pass with 11:25 to play. The Bruins drove to the Arizona State 6 in the waning minutes, but Chris Young sacked Hundley for a 13-yard loss before Fairbairn missed a 37-yard field goal attempt.
UCLA got the ball back at its 35 with 3:21 to play. Evans crossed midfield with a catch on fourth-and-5, but the penalty-prone Bruins committed three fouls on the next two snaps, and Hundley's fourth-down pass to Jordan Payton fell well short of a first down.
The Sun Devils emphatically fought off any flashbacks to the schools' meeting last season, when the Bruins won 45-43 on Fairbairn's 33-yard field goal as time expired.
"I was almost like, 'Oh no, it's happening again,'" Jones said. "But this is a different team. We've trained a lot harder. We're better."
Carl Bradford returned an interception 18 yards for an early score by Arizona State, which racked up 357 yards in a dynamic first half.
The Sun Devils reeled off four touchdowns in the last 16 minutes of the first half, including TD runs by D.J. Foster and Michael Eubank. Bradford made Arizona State's biggest defensive play on the first snap of the second quarter, stepping in front of a short swing pass by Hundley and returning it for a score.
Arizona State drove 62 yards in just 35 seconds during the final minute of the half, with Strong making a 19-yard TD grab with 5 seconds to play for a 22-point lead.


Learn to Connect with Yourself in a World Full of Distractions! - Something New Everyday!
Home happiness Peace Learn to Connect with Yourself in a World Full of Distractions!
Learn to Connect with Yourself in a World Full of Distractions!
What a beautiful quote by Plutarch, one, that couldn't ring more true for the times we live in today. We are constantly bombarded by information and entertainment, be it from the television or the internet. All of this affects us just enough to ensure that the subsequent thoughts we may have feel like we thought of them first.
On a daily basis, we are 'primed' to spend our time or money on something we may not actually need. Consequently, we are always looking forward to the next gadget to purchase, the next movie to watch, or the next television series to enjoy. To top it off, social media has become our new way of connecting with people, enabling us to share everything from pictures of our family to the latest meals we've prepared.
This constant outward search for approval is often the reason why we don't look for an internal source of approval. If on occasion we do look inward, we feel a sense of emptiness and fear. Not knowing what to do with it, we tend to fill the emptiness with an external source of gratification. But this emptiness is important, it tells us that we are disconnected from who we are. And very often, it is this disconnection that ends us up in painful life situations.
But, how can you tell that you have lost connection with yourself? A clear sign is that your true instincts feel like distractions, and distractions feel like your true instincts. When we are distracted, we feel bored, confused and unmotivated. We therefore end up choosing the easiest path from all those available. We become afraid of crying, of getting hurt, of looking at ourselves as we are.
Stop for a moment and notice your surroundings. Do you have a phone or a computer around you? On some level, are you also receiving mental images? Most of the time we are unconsciously seeing things - things we have seen at work, or an image of something our friend sent to us, or even scenes from a favorite TV show. Sometimes, you may see things that you would like to happen, or things that you fear will happen. While the physical eye processes our reality, which is often mundane, the mind's eye shows us a reality which can be quite interesting.
Visualization can often be a double-edged sword. The key to visualization lies in the ability to visualize when we need to and not to when we don't. Otherwise our imagination becomes hyperactive, resulting in a constant stream of images in front of our eyes. It's almost as if we are dreaming, while awake. If we can stop our visualization at will, and only see what our physical eyes are showing us, the mind becomes simplified. It relaxes, drawing our attention inwards, to our bodies. So, our attention moves from things that exist in the mind, to the things which exist in the physical reality.
If someone had to ask you to describe all the emotions you are feeling at this moment, it may be hard to find an answer. More often than not, we experience multiple emotions at the same time. Sometimes we are angry, but at the same time, we may also feel sad because of a life situation. You may also feel at peace, but have a longing for something better. Or you may be full of gratitude, yet not without a hint of pride.
Our body reacts according to our emotions through physical sensations. When our palms sweat, for instance, we know we are nervous. When our heart races, we interpret this as being afraid or excited. When we worry, our muscles become shallow and our muscles and nerves tense up. When we are happy, we breathe easy and our body relaxes.
Our mind, therefore, tells our body what to feel. But we have become used to not paying any attention to what the body is feeling, without this input from the mind. So out of habit, our body obeys our mind. Try this as an example: What are you feeling in the little toe of your left foot? Can you distinguish the sensation in each one of your toes? It's not that easy because our mind has never paid attention to it before.
Try This: Close your eyes and try to discern the shape of your hand, feeling the electrical impulses on the skin and the gentle blood flow in the veins. If you are able to, discern just the index finger or the thumb, try to become more sensitive to what you are feeling until you can feel your entire hand. Within two to five minutes, you will feel your heartbeat and its rhythm pulsating through your hands.
Repeat this exercise with your entire body, helping you develop a full body awareness - a very powerful way of connecting with yourself. Just be careful about negative self-talk if you come across a source of pain, in which you might tell yourself 'Here is that bothersome pain again. What do I do with it?' If you hear negative self-talk such as this, listen calmly. It will point to the next stage of connecting with ourselves.
It is a very normal and common behavior to talk to ourselves. This voice tells us what to do, and what to avoid. Even if we go on a solitary hike in the mountains, hoping to spend time with nature and switch off, we can still hear the 'I' talking. Below is an exercise you can try.
Try This: Close your eyes and pay close attention to whatever sounds there may be around you. Listen to every detail you can hear for thirty seconds, then open your eyes. While doing this exercise, notice, were you talking to yourself? Or did you only experience the act of listening? During this situation, there should be no division, just the pure state of listening, feeling or hearing. In moments where we are fully connected, we become whole. But, what happens when we find the connection? At this moment it is possible to know our pleasures, desires, fears, ambitions, and anxieties for what they actually are. We are able to perceive them with clarity and without any internal conflict.
For example, if fear arises, we may generally notice a couple of things about it. Perhaps you realize that there is nothing dangerous actually taking place, except in our minds. Maybe you notice that the heart rate changes and the muscles tense up as fearful thoughts arise. The voice inside your head may be telling you to be afraid, but in knowing these things we are already one step ahead of fear.
So, the next time it arises, we can predict the pattern, without worrying or over thinking. We will be able to solve the real problem at hand.
In feeling connected, our instincts become stronger. We are able to understand what is right and what is wrong for us. At this point, big decisions can be made easily, without having any regrets. Sometimes, building a strong connection within ourselves is all we need and in some cases, it is all that we have to keep us going.


Cooking Apron "I See Burgers"
Bib apron Burger AP, green with black print, hoop to adjust size, made out of gabardine, weight 240gr.65/35 poly/cotton, dimensions: 70cm width x 92cm height.
Do you love this particular design? Now you can get it printed on your apron so that you can be even more inspired while you cook.


Published on The People's Voice, by Stuart Littlewood, Jan. 2, 2010.
It is desperately sad to see the noble efforts of the Viva Palestina expedition turning sour in front of our eyes as the forces of darkness plot once again to derail humanitarian aid for Gaza.
… Forcing the convoy to take ship and re-route down the East Mediterranean coast through the playground of Israel's marauding gunboats is the sort of lunacy that could have dangerous consequences. Many here are predicting a Dignity-type attack on the ferries that Viva Palestina has been obliged to hire.
I suppose it is a fitting end to a rotten year … a year that began with promise and high hopes but turned out to be one of double-cross and despair. Freedom activist are complaining that all this bickering with Egypt has diverted attention from the main culprits, Israel and America. But other culprits lurk in the evil swamp and Egypt is one of them. It is no bad thing that the spotlight falls on their treacherous behaviour.
So Egypt seeks to legitimise Israel's illegal occupation by colluding with it, and recently has gone further. It is building an Iron Wall to create a hermetically sealed border against the besieged and starving Palestinians, a despicable act that reveals, apart from a cruel streak, an apparent lack of any sense of brotherhood or compassion.
No-one can accuse President Mubarak of being like Macbeth – "too full of the milk of human kindness". (full text).


text:
Q: Compilation when Column doesn't exist I've a Stored procedure which looks like this:
create procedure test as
begin
if  exists(
               select 1 from sys.columns
                where Name      = N'Column2'
                  and Object_ID = Object_ID(Table2')
              )

select Column2 from Table2
end

I want to run this procedure on db where Column2 doesn't exist. I don't want to take existence check out of SP. Currently the error is :

Msg 207, Level 16, State 1, Procedure test, Line
  39 [Batch Start Line 0] Invalid column name 'Column2'.

Is there any way to do so? Why yes and why not?
and why for instance if you check for existence table and select non-existent table that works?

A: Use dynamic SQL:
create procedure test as
begin
    if exists (select 1
               from sys.columns
               where Name = N'Column2' and Object_ID = Object_ID('Table2')
              )
    begin
        exec sp_executesql N'select Column2 from Table2';
    end;
end;

redpajama_set_name: RedPajamaStackExchange


Wednesday's session repeated itself yesterday as investors increased selling in the afternoon session, pushing stocks down on both markets.
On the HCM City Stock Exchange, the VN-Index lost nearly 10 points or 1.58 per cent to close yesterday at 615.80 points.
Blue chips were again the main drags. Among the top 30 shares by market value and liquidity, 22 stocks declined while only six advanced. Gains by Vietcombank (VCB), Masan Group (MSN), Hoang Anh Gia Lai Co (HAG) and Sacombank (STB) helped cushion the decline.
The VN30 fell 0.91 per cent to end at 659.63 points. Oil and gas-related shares ended their winning streak as stocks like PetroVietnam Engineering Consultancy Co (PVE), PetroVietnam Drilling Mud Corp (PVC) and Petrovietnam - Nghe An Investment & Trading Co (PXA) dropped to the floor under heavy selling pressure.
Liquidity remained high with nearly 180 million shares worth VND3.57 trillion (US$169 million) traded by the end of the session.
FLC Group (FLC) secured the position of the most active share on the southern bourse with more than 17 million shares traded, but the share price sank 2.5 per cent to VND11,700 ($0.55) per share.
Analysts at FPT Securities Co said the market was seeing a correction as investors continued to sell shares to realise cash profits after a very long winning streak that sent the VN-Index to a more than six-year high.
Rising bad debts also overshadowed the stock market, with nonperforming loans climbing to 4.11 per cent in July from 3.61 per cent at the end of 2013, according to the State Bank of Viet Nam.
On the Ha Noi Stock Exchange, the HNX-Index also slipped 2.31 per cent to finish yesterday at 88.07 points.
Investors sold securities, oil and gas shares, which showed strong growth in recent months.
Market volume declined 20 per cent from the previous session, totaling nearly 113 million shares, while trading value fell 25 per cent to VND1.72 trillion ($81.5 million).
PetroVietnam Construction (PVX) was again the most active here on trades of 12.4 million shares, but their price dove 4.8 per cent to end at VND6,000 a share.
Foreign investors remained net sellers in HCM City, unloading VND72.3 billion ($6.9 million) worth of shares, but were net buyers in Ha Noi's market, collecting shares worth VND78.3 billion ($3.7 million).


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From hot air ballooning over Montana to nomadic travel in Chile, Brownell can take you where you've never gone before while still providing luxury accommodations and a team of trusted experts to ensure your safety along the way. If hiking and biking is your ideal way to experience nature's beauty, adventure travel is a great way to give you the new perspective you've been craving. Remember, every new quest starts with a leap of faith. Take that leap today and contact your Brownell Advisor to get started planning your next big adventure!


Our workshop is now able to take on any new Barometer repairs.
What Barometers do you service?
We service mainly the Fischer brand but call Cameron if you have another brand you would like repaired.
Barometers are repaired within a 7-10 day period on average depending on the availability of parts.
How do I get it to you? Can I post it to you?
Yes you can either drop of at our workshop or courier to us.
Please make sure that you tell us as much as you can about the issue you need repaired.
Once we receive it we will inform it has arrived and quote on repairs by either by text or email for you to confirm.
How do I get it back? Will you post it back?
Yes we will courier it back to your address.
How will I know when its done?
We will contact you once repair is completed by either phone, cell phone or email.
Why should I choose you for repairs?
We have extensive knowledge of the industry and what is required to complete the repair to a satisfactory condition. Our workmanship is guaranteed.
All of New Zealand for Barometers .
How do I pay for it ?
What courier company do you use? Do I have to sign for it?
We strongly recommend NZ post signature required track & trace courier bags.
How do you package it to ensure it doesn't get damaged?
We Package all jobs as Fragile items with Bubble wrap or re-enforced cardboard.
Do you track and trace?
Yes we use track and trace with new post or pack & send with Castle Freight.
All our workmanship is cover by our 1 year warranty.


text:
Ganda Singh (15 November 1900 – 27 November 1987)  was a Punjabi historian and Padma Bhushan awardee. In addition to scores of research papers, booklets and pamphlets, he published over two dozen full-length volumes of historical value.

Early life
Dr. Ganda Singh was born on 15 November 1900, at Hariana, a town in Hoshiarpur district of  British Punjab. He was schooled in the local Government Middle School and then the D.A.V. Middle School. He matriculated from Government High School, Hoshiarpur. Then he went to the Forman Christian College, Lahore.

Researcher and historian
In October 1931 began Dr Ganda Singh's long and fruitful career as a researcher and historian. The Khalsa College, Amritsar placed him in charge of its newly created Sikh History Research Department, a position he kept till 1949. During this period he travelled extensively, rummaging various public libraries, archives and private collections throughout India in quest of materials on Sikh history, enriching the library of his department. He also brought out several books and articles based on these.

In 1938, he had been appointed a corresponding member of Indian Historical Records Commission of Government of India, and was a full member of the Commission from 1950 to 1956.

In 1949, he was appointed Director of Archives and Curator of Museum under the Government of Patiala and East Punjab States Union. In 1950 he received the additional charge of Director of the Punjabi Department. His thesis on Afghan warlord Ahmad Shah Durrani earned him the degree of Doctor of Philosophy from the Punjab University, Chandigarh, in 1954, as well as much applause from scholars and historians, including  Maulana Abul Kalam Azad.

Dr Ganda Singh was principal of the Khalsa College, Patiala, when he was invited by the Punjabi University, Patiala, to organize its Department of Punjab Historical Studies. in 1965, he set up Punjab History Conference. In 1967, he launched the University's journal, the biannual The Panjab Past and Present, of which he was the editor.

He was nominated member of Punjab Regional Committee for the Survey of Historical Records, Government of Punjab, Lahore, and of Indian Historical Records Commission, Government of India. He was secretary of the Committee for the History of Freedom Movement in PEPSU, Patiala, and chairman of the Regional Records Survey Committee for History of Freedom Movement, Shimla/Patiala, from November 1957 to December 1962. He held membership of The Asiatic Society, Calcutta, Indian Institute of Historical Studies, Calcutta, Royal Asiatic Society of Great Britain and Ireland and Bharat Itihas Sanshodhak Mandal, Pune. He presided over the medieval section of Punjab History Conference session of the Institute of Historical Studies, Calcutta, held at Shillong in 1974 as well as over its 13th session held at Panaji (Goa) in 1975. In 1974, he presided the 35th session of Indian History Congress at Jadavpur.

Awards
The Government of India honoured him with Padma Bhushan award in 1984.

He had two honorary degrees of Doctor of Letters awarded to him, by Aligarh Muslim University on 19 December 1964 and by Punjabi University on 25 February 1978.

The Punjab Government invested him with the Award for Literature on 31 March 1963. On 28 March 1964, the Shiromani Gurdwara Parbandhak Committee officially commended him. "Sikh Educational Conference" honoured him during its 52nd annual session held at Kanpur on 25–27 October 1974, and Punjabi University, Patiala, at the annual session of the Punjab History Conference held during November 1976. The University also brought out during the same year an anthology, Essays in Honour of Dr Ganda Singh, edited by his old pupil Professor Harbans Singh. The Indian History Congress during its Silver Jubilee session held at Panaji (Goa) on 27 November 1987 honoured him as one of the five distinguished historians of India.

Death
Dr. Singh died at Patiala on 27 November 1987.

Legacy
He donated his entire personal collection of rare books, maps, documents and manuscripts, which occupied several rooms of his modest residence on the Lower Mall at Patiala, to Punjabi University, Patiala.

References

1900 births
1987 deaths
People from Hoshiarpur district


text:
Twitter announces acquisition of CardSpring
Twitter has recently announced another acquisition. According to an article in Tech Crunch, the social media site has purchased CardSpring, a company that creates apps for payment cards, to enable ecommerce through coupons and other services like loyalty cards. It works by making it easier for developers to create these offers.
CardSpring's services allow developers to "link digital applications to credit or debit cards," according to a description on the company's website. An acquisition may present an opportunity for the company being acquired to expand its technologies, as it will be increasing its resources by joining forces with a larger business.
"When we started talking with the Twitter commerce team, we quickly realized our philosophies were perfectly aligned, and by joining them, we'd be able to significantly accelerate our vision," said CardSpring on its website.
For Twitter, this acquisition strategy could help the company provide increased value to its service by offering customers the opportunity to easily take advantage of deals as they use the site. This way, Twitter looks to possibly delve deeper into the ecommerce landscape.
A Bloomberg article details that Facebook is also looking to make ecommerce accessible through its network and the company is testing a "buy" button to allow users make purchases. With the acquisition of CardSpring, Twitter looks to position itself to be a stronger competitor in the marketplace.
It has been discussed previously on this blog that as ecommerce continues to grow, mobile is playing an important role as customers use their mobile devices to socialize on sites like Twitter and Facebook. The integration of ecommerce features could potentially help these companies generate additional revenue.
Companies seeking to develop an acquisition strategy can consult an M&A advisor for expert advice.
« Yahoo acquires Flurry
NetSuite acquires Venda for e-commerce capabilities »


text:
Q: How can i not repeat this function I have a filtering system on my blog, it works fine, but im just wondering how i can not repeat this function over and over.
heres the html:

all

another link

third link

fourth

fifth

and the jquery i am using:
$('.filter1').on('click', function(e){
    e.preventDefault();

    // add & Remove filter

    $('.filter').removeClass('filterActive');
    $('.filter1').addClass('filterActive');

    // hide & show categories

    $('.categ').hide();
    $('.categ1').show();
});

$('.filter2').on('click', function(e){
    e.preventDefault();

    // add & Remove filter

    $('.filter').removeClass('filterActive');
    $('.filter2').addClass('filterActive');

    // hide & show categories

    $('.categ').hide();
    $('.categ2').show();
});

i repeat this 5 times, i know what i want to do, i want to see if the filter clicked is equal to 1,2,3,4,5 then show the code for the responding function. but im not sure where to start.
Forgot to mention i have tried this:
var filterNum;

$('.filter').on('click', function(e){
    e.preventDefault();

    if($('.filter').hasClass('filter1')){
        filterNum = 1;
    }else if($('.filter').hasClass('filter2')){
        filterNum = 2;
    }else if($('.filter').hasClass('filter3')){
        filterNum = 3;
    }else if($('.filter').hasClass('filter4')){
        filterNum = 4;
    }else if($('.filter').hasClass('filter5')){
        filterNum = 5;
    }

    filterClick(filterNum);
});

function filterClick(number){
    // add & Remove filter

    $('.filter').removeClass('filterActive');
    $('.filter' + number).addClass('filterActive');

    // hide & show categories

    $('.categ').hide();
    $('.categ' + number).show();
}

A: var $filters = $('.filter');
var $filtered = $('.categ');

$filters.on('click', function(){
   $filters.removeClass('filterActive');
   $(this).addClass('filterActive');
   $filtered.removeClass('filterActive');
   $filtered.eq(k).addClass('filterActive');
});

https://codepen.io/mrbizle/pen/vZKYgq
This way we don't have to create loads of jQuery objects

A: Use this to refer to the clicked elemten. Additionally to that you can use a custom data attribute to reference to the categories you want to show:

$('.filter').on('click', function(e) {
  e.preventDefault();

  // add & Remove filter
  var $this = $(this);

  $('.filter').removeClass('filterActive');
  $this.addClass('filterActive');

  // hide & show categories
  $('.categ').hide();
  $('.' + $this.data('cat')).show();
});
.filterActive {
  color: red;
}

all

another link

third link

fourth

fifth

    Cat1

    Cat2

    Cat3

    Cat4

    Cat5

A: If you can modify the html, I would use data-attributes to make this easier:

all

another link

third link

fourth

fifth

$('.filter').on('click', function(e){
    e.preventDefault();

    $('.filter').removeClass('filterActive');
    $(this).addClass('filterActive');

    $('.categ').hide();
    $('.categ' + $(this).data('filterid')).show();
});

A: Where is this .categ? Assuming that it's inside the
, this should work:

$('.filter').on('click', function(e) {
  e.preventDefault();
  // add & Remove filter
  $('.filter').removeClass('filterActive');
  $(this).addClass('filterActive');
  // hide & show categories
  $('.categ').hide();
  $(this).next('.categ').show();
  /* If .categ is inside the .filter, use this instead:
    $(this).find('.categ').show();
     And if .categ is outside, you'll need to use something like:
    $(this).parents().next('.categ').show();
  */
});

allcateg

another linkcateg

third linkcateg

fourthcateg

fifthcateg


text:
Роберт Элмер Мартин (, 19 февраля 1910 — 1978) — канадский шахматист. Несмотря на достаточно редкое участие в соревнованиях, добивался серьезных успехов и за доской, и в заочной игре. В 1933 г. выиграл чемпионат Канады. В 1946 г. стал победителем чемпионата Канады по переписке. В период с 1930 по 1940 гг. трижды выигрывал чемпионат Торонто. В составе сборной Канады участвовал в международном матче по радио против сборной команды Австралии (выступал на 7-й доске, завершил свою партию вничью).

Спортивные результаты

Примечания

Ссылки
 Партии Р. Мартина в базе Chessgames
 Личная карточка Р. Мартина на сайте 365Chess

Шахматисты Канады
Шахматисты XX века


text:
You are at:Home»Movie Reviews»Review: 'Steve Jobs: The Man in the Machine', a Film by Alex Gibney
Review: 'Steve Jobs: The Man in the Machine', a Film by Alex Gibney
By Mae Abdulbaki on	 September 2, 2015 0 Comments
Award-winning filmmaker Alex Gibney has always been about uncovering the truth behind the subjects of his documentaries. Always blunt and always willing to get the inner workings of anything or anyone he chooses to spotlight instead of glorifying anything, it is with intense fervor that he showcases the former head of Apple in Steve Jobs: The Man in the Machine in a balance, yet gray light and wonders why, in a world where so many people made famous by one thing or another, the world mourned Steve Jobs differently.
Steve Jobs was many things to many people: innovator, tech pioneer, manipulator, formidable, husband, father, friend, and enemy. And all these adjectives used to describe Jobs are brought to light in a way that the 2013 film Jobs, where Ashton Kutcher played the computer mogul, didn't. Practically a living legend during his lifetime, Jobs founded Apple, Inc. in 1976 with Steve Wozniak and Ronald Wayne as a way to sell and innovate the personal computer, which at that point wasn't something anyone had really paved the way to do. The film goes through the ups and downs of his professional and personal life up until his death in 2011.
Directed and narrated by Alex Gibney, The Man in the Machine (the narrative drama, titled Steve Jobs and starring Michael Fassbender will be out later this fall) does a wonderful job in the taking a road less traveled by when documenting a public figure. The film is thought-provoking and unabashed in both its obvious admiration of Jobs and the struggle to come to terms with the fact that he wasn't always the most genial of men to work with or be around. The man was a workaholic and his work took precedent over everything else in his life. Jobs goes so far as to disavow his ex and daughter, making up rumors and claiming that he isn't the father to his first-born Lisa (of which he later named a computer model after).
But within that man was also a pioneer who paved the way for the personal computer and of course later, the smart phone. So there are two sides of the coin: brilliance and brutality. Gibney explores whether or not the general public loved him personally or just the products Jobs and Apple made for them. And the film leans into the smoke that sometimes covers up people in their public personas that we often find ourselves ignoring because we "like" the person. Or what the person shows us. These questions and the depth of Gibney's analytical take on Jobs as a person and businessman is what makes for a strong film. You'll find yourself on both sides of the spectrum. One moment you're hating Jobs and at another moment appreciating his brilliance and dedication to his craft and company.
Steve Jobs: The Man in the Machine is an honest film about the life of Jobs. Using archive footage and interviewing the people who were closest to him, the film removes the rose-colored glasses to bring us the real man behind the technology that we are so very fond of today. It's filled with firs-person accounts of unbelievable things that happened during the span of his life. Many of them aren't very nice things and reflect the kind of man Jobs could be, versus the creative and work-obsessed guy that could be called too passionate. Gibney isn't afraid to delve into Jobs's darker side and the film is thankfully not praising Jobs and glossing over important knowledge that so many other documentaries can do. The film is irrevocable one of the strongest documentaries to come out this year.
Release Date: September 4, 2015 | Director and Screenwriter: Alex Gibney | Genre: Documentary | MPAA Rating: R for some language
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Mae Abdulbaki
Mae is a Washington, DC-based film critic, entertainment journalist and Weekend Editor at Heroic Hollywood. A member of the Washington, DC Area Film Critics Association (WAFCA), she's a geek who loves discussing movies and TV. She is also a voting member of the Black Reel Awards. If she's not at the movies, she's catching up on her superhero TV-watching, usually with a glass of wine in hand.


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