Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.
Previtera, M. L., Langrana, N. A. Preparation of DNA-crosslinked Polyacrylamide Hydrogels. J. Vis. Exp. (90), e51323, doi:10.3791/51323 (2014).
Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.
Static and dynamic substrates are two categories of biomaterials that were developed to study the effects of tissue elasticity or stiffness on cell function. Static substrates are unable to change their physical properties after they are fabricated and/or once cells are plated. Polyacrylamide (PA) gels were the first two-dimensional, static substrates that were synthesized for mechanobiology investigations 5,17. PA gels are easy to prepare, inexpensive, versatile, and can be fabricated with a broad range of elastic moduli. Although these technical advantages make PA gels a commonly applied substrate, static substrates are not indicative of the dynamic nature of the extracellular matrix (ECM) and surrounding cellular environment in vivo. For example, the ECM undergoes stiffness alterations as a result of injury, development, or disease. Dynamic substrates are therefore favored as tissue-mimicking substrate models in mechanobiology studies 22,24,25.
Numerous synthetic, natural, two-dimensional, three-dimensional, static, and dynamic biomaterials have been developed to mimic tissue stiffness 1,3,6,16,23,26. Some dynamic substrates require heat, UV, electrical current, ions, and pH changes to alter their mechanical properties 2,4,7,8,12,15,16, but these stimuli can restrict the hydrogel's bio-application. DNA-crosslinked polyacrylamide hydrogels (DNA gels) are dynamic two-dimensional elastic substrates. DNA crosslinks allow for temporal, spatial, and reversible modulation of DNA gel stiffness by addition of single-stranded DNA (ssDNA) to media or buffer 9-11,13,14,18,21. Unlike the aforementioned dynamic gels where stimuli are applied for modulation of elasticity, the DNA gels rely on the diffusion of applied ssDNA for the alteration of elasticity. Therefore, the upper gel surface, where cells are grown, is the first area modulated because the rate of elasticity modulation is dependent on the gel thickness.
DNA gels are similar to their PA gel counterparts in that they have a polyacrylamide backbone, however the bis-acrylamide crosslinks are replaced with crosslinks composed of DNA (Figure 1). Two ssDNAs (SA1 and SA2) hybridize with a crosslinker strand (L2) to make up the DNA crosslinks of the gel. SA1 and SA2 have distinct sequences that both contain an Acrydite modification at the 5´ end for effective incorporation into the PA network. For preparation of the gels, SA1 and SA2 are individually polymerized into a PA backbone and, subsequently, the polymerized SA1 and SA2 are mixed together. L2, the crosslinker, is added to the SA1 and SA2 mixture. The L2 base sequence is complementary to both SA1 and SA2 sequences and L2 hybridizes with SA1 plus SA2 to form the DNA crosslinks. Initial, DNA gel elasticity is determined by both L2 concentrations and crosslinking (Tables 1 and 2). DNA gels containing equal stoichiometric amounts of L2, SA1, and SA2 are the stiffest gels because SA1 and SA2 are 100% crosslinked by L2 (designated as 100% gels). Lower concentrations of L2 result in a lower percentage of DNA crosslinking and, therefore, softer DNA gels. Gels as low as 50% crosslinked (designated as 50% gels) have been constructed 9-11.
Figure 1. DNA gel crosslinking and uncrosslinking schematic 9-11,13,14,18,21. Step 1: SA1 (red) and SA2 (blue) are individually polymerized into a polyacrylamide backbone (black). After polymerization, SA1 and SA2 polymerized solutions are mixed together. Step 2: L2 (green) is added and hybridizes with SA1 plus SA2 to form the crosslinks of the gel. Step 3: R2 hybridizes with the toehold of L2. Step 4: Toehold hybridization of R2 propels the unzipping of L2 from SA1 and SA2.
Unlike PA gels, DNA gels can stiffen and soften after synthesis. For that reason, cells grown on DNA gels can be subjected to dynamic stiffness alterations. To stiffen cell-adherent gels, L2 can be added to the culture media of low percentage gels to increase the percentage of crosslinks. To soften cell-adherent gels, L2 can be removed to decrease the percentage of crosslinks 10,13,21. L2 has an additional toehold sequence at the 3´ end to allow L2 to uncrosslink from SA1 and SA2 (Table 1). Removal of L2 is accomplished by hybridization of a reversal strand called R2. R2 is complementary to the full length of L2 and hybridizes first with the L2 toehold. Toehold hybridization propels the unzipping of L2 from SA1 and SA2, which eliminates the crosslink and reduces the gel stiffness.
In this report and video, step-by-step instructions are provided for the preparation of stiffening and softening DNA gels. While 100% and 80% gel preparations are described, this protocol can be tailored to create DNA gels of other initial and final crosslinked percentages. In general, 100% and 80% gels are prepared, immobilized onto glass cover slips, functionalized, and seeded with cells. L2 is added to the media of 80% gels and R2 is added to the media of 100% gels, 48 hr after plating. The addition of L2 to media stiffens 80% gels to 100% crosslinked, whereas the addition of R2 to media softens 100% gels to 80% crosslinked. Stiffened gels are designated as 80→100% gels and softened gels are designated as 100→80% gels in the text. For control or static gels, ssDNA consisting of Ts or As is delivered to another set of 100% and 80% gels. After a minimum of two days following elasticity modulation, cells can be processed and analyzed.
L2 30 TCA TTC TGA CGC AAA GGT GCG CTA CAC TTG 56 A 10 bp toehold sequence is included.
L2 40 TCT GAT TGG GAA ACA GTC CTA TGC CAC GGT TAC CTT CAT C 65.9 A 12 bp sequence toehold is included.
L2 40 TCC AGT CGT CCC TAA GCA TGG ACA TTG CAT ACA CCT CCG T 68.8 Toehold is not included.
Table 1. Base sequences for ssDNA 9-11,13,14,18,21. Cellular and mechanical studies have utilized several different crosslink designs to generate DNA gels with a range of static and dynamic mechanical properties. The parameters modulated in crosslink design are base sequence and sequence length or crosslink length. Bold and italicized fonts illustrate base pairing between SA1 and L2 and between SA2 and L2, respectively.
Table 2. Young's modulus (E) of DNA gels 9-11,13,14,18,21. Acrylamide concentration, crosslink percentage, and crosslink length can be modulated in DNA gels. Designs 1, 2, and 3 have 20, 28, and 40 bp crosslink lengths, respectively. 100% gels for all designs have similar moduli indicating crosslink length does not affect gel elasticity. However, variations in acrylamide concentration alter DNA gel elasticity.
NOTE: The entire protocol from gel preparation to cell processing takes a minimum of six days. Estimated time for gel preparation is 8 hr plus an O/N incubation. Estimated time for gel immobilization and DNA annealing is 8 hr plus an O/N rinsing step. Estimated time for gel functionalization is 2 hr. Time for cell plating and growth is dependent on culture type and application, but a minimum of four days is required.
NOTE: Prepare DNA gels in three distinct steps. First, individually polymerize SA1 and SA2 ssDNA into a PA backbone. These solutions are called SA1 polymerized solution and SA2 polymerized solution, respectively (§1.1). Second, dissolve the crosslinker, reversible strand, and control ssDNAs. Dissolve the lyophilized L2 ssDNA (crosslinker). This is called 100% L2 solution and is used to fabricate 100% gels (§1.2). Dilute an aliquot of 100% L2 solution to 80%. This is called 80% L2 solution and is used to fabricate 80% gels. Dissolve lyophilized R2 (reversible strand) and control ssDNA to use in §4. Third, mix SA1 polymerized solution, SA2 polymerized solution, and 100% or 80% L2 solution in a ratio of 10:10:6 (SA1:SA2:L2) to form 100% and 80% gels, respectively. (§1.3).
Select and order the appropriate SA1, SA2, L2, and R2 base sequences from Tables 1 and 2. Base sequences and sequence length were optimized in previous reports 9-11,13,14.
Calculate all solution formulations (Tables 3-4). NOTE: Meticulously document calculations for troubleshooting purposes. Construction of an Excel template is recommended and can be used repeatedly for DNA gel preparation. Please see Tables 3 and 4 for an example of the tabulation for Design 2 (Tables 1 and 2). The volumes specified in Table 4 will be used throughout this protocol but volumes will vary with every aliquot of lyophilized ssDNA.
Table 3. Percentage of solutions for fabricating SA1 or SA2 polymerized solutions. The first column shows the solutions for formulating the DNA gels. The second column shows the stock concentrations of these solutions. The third column shows the percentage of the stock solutions in SA1 or SA2 polymerized solutions (v/v). The last column reflects the final concentrations in SA1 and SA2 solutions.
SA1 solution 320.7 nmol of lyophilized SA1 ssDNA 3.00 mM 320.7 nmol / 3.00 nmol μl-1 = 107 µl 107 µl of TE buffer to lyophilized ssDNA SA1 solution is 60% of SA1 polymerized solution.
178 µl is the total volume of SA1 polymerized solution (Table 3).
SA2 solution 324.4 nmol of lyophilized SA2 ssDNA 3.00 mM 324.4 nmol / 3.00 nmol μl-1 = 108 µl 108 µl of TE buffer to lyophilized ssDNA SA2 solution is 60% of SA2 polymerized solution.
180 µl is the total volume of SA2 polymerized solution (Table 3).
SA1 polymerized solution 40% acrylamide 10% 178 µl x 0.25 = 44.5 µl 45 µl Calculate the amount of acrylamide, TBE, APS, and TEMED based on a total volume of 178 µl (see above and Table 3).
100% SA1 solution 60% — 107 µl SA1 solution is 60% of the SA1 polymerized solution (see above and Table 3).
2% APS 0.05% 178 µl x 0.025 = 4.45 µl 4.5 µl Add and mix APS before adding TEMED.
SA2 polymerized solution 40% acrylamide 10% 180 µl x 0.25 = 45 µl 45 µl Calculate the amount of acrylamide, TBE, APS, and TEMED based on a total volume of 180 µl (see above and Table 3).
100% SA2 solution 60% — 108 µl SA2 solution is 60% of the SA2 polymerized solution (see above and Table 3).
100% Gel solution 100% SA1 polymerized solution 10 parts — 10 µl Compose 100% gels with the following SA1:SA2:L2 ratio, 10:10:6.
80% Gel solution 100% SA1 polymerized solution 10 parts — 10 µl Compose 80% gels with the following SA1:SA2:L2 ratio, 10:10:6.
80→100% gel 80% gel 100% gel Calculation 1: 1 µl of 100% L2 solution into culture media Calculations are based on having 20 µl gels on cover slips. First, convert the parts of 100% L2 solution in the 20 µl of gel into µl. Second, calculate the amount (in µl) of 100% L2 solution needed for an additional 20% of crosslinking. Add this amount of 100% L2 solution to gel.
100→80% gel 100% gel 80% gel Calculation 1: 1 µl of 100% R2 solution into culture media Calculations are based on having 20 µl gels on cover slips. First, convert the parts of 100% L2 solution in the 20 µl of gel into µl. Second, calculate the amount (in µl) of 100% L2 solution needed to be removed to compose a 20% gel. Add this amount of R2 solution to gel.
100% gel (Control) 100% gel 100% gel — 1 µl of control solution into culture media Amount of control solution is equivalent to the amount of R2 solution added.
80% gel (Control) 80% gel 80% gel — 1 µl of control solution into culture media Amount of control solution is equivalent to the amount of 100% L2 solution added.
Table 4. Example calculations for DNA gel preparation. Mock numbers are provided to illustrate the calculations for the preparation of 80%, 100%, 100→80%, and 80→100% gels. DNA gels are Design 2 in Table 1.
Centrifuge lyophilized SA1 ssDNA at 2,000 x g for 15 sec to ensure all ssDNA is at the bottom of the vial. NOTE: Proper concentration of SA1 solution is critical for DNA gel synthesis.
Prepare 3 mM of SA1 solution by adding 107 µl of 1x Tris-EDTA, pH 8.0 buffer (TE buffer) to the vial (Tables 3-4).
Heat SA1 in TE buffer at 70 °C for 5 min or until ssDNA pellet is completely dissolved. NOTE: Heating temperature should be a minimum of 5 °C above melting temperature (Tm) to ensure DNA is in a single-stranded state.
Prepare SA1 polymerization solution by adding 40% acrylamide and 10x Tris-Borate-EDTA (TBE) buffer at the indicated percentages (Table 3). In this example, add 45 and 18 µl, respectively, to 107 µl of SA1 solution (Table 4).
Degas with nitrogen gas for 3 min to facilitate mixing. Insert a p200 tip attached to a nitrogen gas source into the solution and slowly allow nitrogen gas to pass through the solution. Test the flow rate of nitrogen gas in water prior to degassing SA1 solution to prevent splatter.
Centrifuge for 15 sec at 2,000 x g to gather solution.
Add 4.5 µl of 2% ammonium persulfate (APS, Tables 3-4) to initiate gel polymerization.
Invert the tube several times to mix.
Centrifuge for 15 sec at 2,000 x g to gather solution for homogenous polymerization.
Add 4.5 µl of 20% tetramethylethylenediamine (TEMED, Tables 3-4) to catalyze polymerization.
Degas with nitrogen gas for 3-5 min to complete polymerization and minimize un-reacted monomers.
Incubate solution for 10 min at RT to complete polymerization. NOTE: This solution is called SA1 polymerized solution.
Repeat steps 1.1.3-1.1.16 with lyophilized SA2 ssDNA, but add 45, 18, 4.5, and 4.5 µl of acrylamide, TBE, APS, and TEMED, respectively, to 108 µl of SA2 solution (Tables 3-4). NOTE: SA1 and SA2 polymerized solutions can be stored at 4 °C for up to one month.
Repeat steps 1.1.3-1.1.5 with lyophilized R2 ssDNA but add 64.4 µl of TE buffer (Table 4).
Repeat steps 1.1.3-1.1.5 with lyophilized control ssDNA but add 111 µl of TE buffer (Table 4).
Repeat steps 1.1.3-1.1.5 with lyophilized L2 ssDNA but add 219 µl of TE buffer (Table 4). NOTE: This is 100% L2 solution. Adding 100% L2 solution to SA1 and SA2 polymerized solutions (see §1.3) will form a 100% crosslinked DNA gel (100% gel) because SA1, SA2, and L2 will be stoichiometrically equivalent.
Dilute an aliquot of 100% L2 solution to 80% by adding 20 µl of 1x TE buffer to 80 µl of 100% L2 solution (Table 4). NOTE: This solution is 80% L2 solution. Adding 80% L2 solution to SA1 and SA2 polymerized solutions (see §1.3) will form an 80% crosslinked DNA gel (80% gel) because only 80% of SA1 and SA2 will be crosslinked to L2. Solutions can be stored at 4 °C for up to one month.
Heat SA1 and SA2 polymerized solutions at 70 °C until solution viscosity is reduced (about 1 min). Raise the temperature up to 80 °C if solution is still too viscous to pipette.
Add 10 µl or 10 parts of SA1 polymerized solution to 10 µl or 10 parts of SA2 polymerized solution (Table 4). NOTE: SA1 and SA2 polymerized solutions are extremely viscous and difficult to pipette. Since concentrations are critical to gel formation, use a positive-displacement pipette from this point forward or see discussion for other handling techniques. Also, pipette in multiple, small aliquots (>20 µl) rather than a single, large volume.
Mix SA1 and SA2 polymerized solutions together via alternating heating (for 15 sec at 70 °C) and stirring (for 15 sec with a pipette tip) for a total of 1 min.
Add 6 µl or 6 parts of 100% L2 solution to form a 100% gel (Table 4).
Mix by alternating heating and stirring as described in step 1.3.3.
Heat gel for 1 hr at the optimal annealing temperature (35 °C). Calculate annealing temperature by subtracting 5 °C from the ssDNA sequence with the lowest melting temperature (Table 1).
Pipette the 100% gel into a 60-mm Petri dish.
Allow DNA crosslinks to continue to rehybridize by incubating gel for 4 hr at RT.
Cover gel with PBS containing calcium and magnesium and incubate O/N at RT. NOTE: Gels will swell about 3 times their initial volume. In addition, gels will equilibrate with buffer and excess acrylamide will diffuse out of the gels. Calcium and magnesium in the PBS stabilize DNA hybridization and prevent gel bursting (see Discussion for toubleshooting gel bursting).
Remove remaining buffer from Petri dish.
To fabricate 80% gels, repeat steps 1.3.1-1.3.10 but replace 100% L2 solution in step 1.3.4 with 80% L2 solution. Store 100% and 80% gels at 4 °C for up to one month. NOTE: The stiffness differences between 80% and 100% gels are physically distinguishable.
NOTE: Immobilize aliquots of 100% or 80% gels onto glass cover slips (Figure 2).
Heat 100% gel at 70 °C for about 30 sec or until gel is less viscous for pipetting.
Place glass cover slips onto a 70 °C heat block and allow to heat for 1-2 min.
Add a drop of optical adhesive to each glass cover slip.
Add 20 µl of 100% gel to each glass cover slip (Table 4). NOTE: 10-30 µl can be dispensed on each cover slip.
Allow gel to melt and spread over glass cover slip. NOTE: If gel topology does not appear even after melting, flatten gel with a siliconized glass cover slip. However, additional swelling will occur in subsequent steps and contribute to gel unevenness.
Remove glass-containing gel from heat block and place into a 24-well tissue culture dish.
Repeat steps 2.1-2.6 with 80% gel.
Heat gels for 1 hr at the optimal annealing temperature (35 °C). NOTE: It is normal for gels to become dry and this will be resolved when gels are incubated in PBS.
Expose plates containing 80% and 100% gels to ultraviolet (UV, 365 nm) light for 15 min. NOTE: UV exposure simultaneously cures glue and sterilizes gels. If a UV crosslinking chamber is not available, gels can be placed under a UV light that is equipped in a biological safety cabinet. After UV exposure, perform subsequent steps of this protocol under a biological safety cabinet to maintain gel sterility. Also, use sterile solutions from this point forward.
Allow DNA crosslinks to rehybridize at RT for 4 hr.
Add PBS and incubate O/N at 4 °C. NOTE: PBS incubation rinses, equilibrates, and swells gels again because repeated heating can dehydrate the gels.
Figure 2. Schematic of gel immobilization and functionalization. After DNA gels (grey) are prepared, gels are attached to glass cover slips (white) by optical glue (green). Gels are simultaneously cured and sterilized by UV light. After swelling, gels are approximately 1-mm thick. Next, gels are functionalized in a two-step process (red outlined box). First, sulfo-SANPAH is conjugated to acrylamide on the gel surfaces by UV light exposure. Second, gels are incubated with collagen or poly-D-lysine, which attaches to the sulfo-N-hydroxysuccinimide ester in sulfo-SANPAH. This figure has been modified from 10.
NOTE: DNA gel functionalization is required for cell adhesion since polyacrylamide gels are inert materials (Figure 2). In this section, gels will be functionalized in two-steps. First, N-Sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate or sulfo-SANPAH will be covalently linked by photolysis of nitrophenyl azide group to the polyacrylamide backbone of the DNA gels. Second, primary amines in collagen or poly-D-lysine will attach to the sulfo-N-hydroxysuccinimide ester in sulfo-SANPAH to attach the proteins to the gel surface.
Remove buffer in wells with a pipette and be careful not to aspirate the gel. NOTE: Do not perform vacuum aspiration to reduce the probability of aspirating the gel.
Optional) Wash gels thrice for 15 min at RT to remove excess acrylamide monomer, TEMED, and APS.
Add about 300 µl of sulfo-SANPAH to cover each gel.
Expose gels to UV (365 nm) for 5 min.
Rinse gels once with PBS to remove excess sulfo-SANPAH.
Incubate gels in about 300 µl of 0.2 mg/ml of poly-D-lysine for 1 hr at RT or O/N at 4 °C. NOTE: Alternatively, incubate gels with 0.2 mg/ml of collagen type 1 O/N at 4 °C.
Wash gels twice with PBS for 5 min to remove excess poly-D-lysine.
Incubate gels in about 300 µl of media for 30 min to equilibrate gels.
Remove media immediately before plating cells.
NOTE: In this section, we provide details regarding the softening or stiffening of gels after cell plating. A detailed cell culturing protocol is not provided for several reasons. First, numerous cell types can adhere onto DNA gels and each cell type will require specific adjustments by the researcher for cell plating. Second, standard cell and tissue culturing techniques are used for these experiments and can be found in numerous original articles, technical articles, and reference books. Techniques for specifically plating fibroblasts and neurons onto DNA gels can be found in previous publications 9-11,19-21.
Plate cells. For protocols regarding dissection and/or culturing of neurons or fibroblasts mentioned in this article, refer to one of the following publications 9-11,19-21.
After a minimum of 48 hr, modulate gel stiffness by pipetting 1 µl of control, L2, or R2 solution close to the gel (Table 4).
To stiffen gels from 80% to 100% crosslinked (80→100% gels), add the remaining 20% of L2 to 80% gels by adding 1 µl of 100% L2 solution close to the gel.
To soften gels from 100% to 80% crosslinked (100→80% gels), add R2 to remove 20% of the crosslinks from 100% gels by adding 1 µl of 100% R2 solution close to the gel.
For controls, add equal volumes of control solution to a subset of 100% and 80% gels by adding 1 µl of control solution close to the gel.
Perform the desired cell processing and data analysis after a minimum of 48 hr.
Prior to our studies, cell-ECM interactions were observed on static compliant biomaterials or on irreversible and unidirectional dynamic substrates. These substrates do not accurately reflect the dynamic nature of the cellular microenvironment. Our work shifts the existing technical paradigms by providing a more physiologic model for studying cell-ECM interactions on softening and stiffening dynamic biomaterials. In previous studies, we analyzed the effects of dynamic substrates on cells by examining various cell morphologies and phenotypes on these gels. In one example, we determined the effects of substrate stiffening on fibroblasts by evaluating fibroblast morphology on dynamic DNA gels. L929 cells, a mouse-derived fibroblast cell line, were plated on 80% and 100% gels (Figure 3) 11. Two days after plating, 20% additional L2 solution was added to media of 80% gels to stiffen the gels to 100% crosslinked (80→100%, center panel). 100% gels and another set of 80% gels received control ssDNA to remain static (right and left panel, respectively). Light microscopy images were captured two days after delivery of DNA to the media. Image blurriness, as seen in Figure 3, was due to gel surface unevenness and is typical and unavoidable. Gel swelling and contraction, resulting from the addition of buffers and L2 18,21, contributes to gel unevenness. Morphology was evaluated with ImageJ software (NIH, Bethesda, MD) and served as an indicator of function. Fibroblasts grown on stiffening gels (80→100%) had no alterations in aspect ratio, but exhibited a larger projection area than fibroblasts grown on 100% gels 11. Interestingly, the morphology of fibroblasts grown on static gels was dissimilar to the morphology of fibroblasts grown on dynamic gels. Fibroblasts grown on 100% gels had a smaller projection area and larger aspect ratio than fibroblasts grown on 80% gels. These results indicate that fibroblast behavior is dependent on the dynamic nature of the microenvironment. In another study, the effects of substrate softening on neurons was determined by analyzing neuronal phenotypes including dendrite number, dendrite length, and axon length (Figure 4) 10. Rat-derived spinal cord neurons were grown on 100% and 50% gels. Four days after plating, 50% R2 was added to the media of 100% gels to soften the gels to 50% crosslinked (100→50%, center panels). 50% gels and another set of 100% gels received control ssDNA to remain static (right and left panels, respectively). After three days, neurons were fixed and immunostained with a dendritic marker (MAP2, top panels), an axonal marker (Tau, middle panels), and nuclei (DAPI, bottom panels) to evaluate phenotypes. Neurons grown on softening gels (100→50%) had no differences in primary dendrite number or axon length, but exhibited shorter primary dendrites than neurons grown on 50% gels. As in fibroblast studies, neural phenotypes on static gels were dissimilar to neural phenotypes on dynamic gels. Neurons grown on 50% gels had fewer primary dendrites and longer axons than neurons grown on 100% gel. In conclusion, fibroblast and neuron images demonstrate that multiple cell types can be studied on dynamic and static DNA gels, DNA gel unevenness causes imaging challenges, standard morphological and immunostaining techniques are feasible, and most importantly the behavior of cells is dependent on substrate dynamics.
Figure 3. Fibroblasts grown on static and dynamic DNA gels. Representative light microscopy images of L929 fibroblasts grown on 80% (left panel), 80→100% (center panel), and 100% gels (right panel). Scale bar is 100 μm. This figure has been modified from 11. Please click here to view a larger version of this figure.
Figure 4. Neurons grown on DNA gels. Representative fluorescent images of spinal cord neurons grown on 100% (left panels), 100→50% (center panels), and 50% (right panels) gels. MAP2 immunostaining indicates dendrites (top panels), Tau-1 immunostaining indicates axons (middle panels), and DAPI staining indicates nucleus (bottom panels). Scale bar is 100 μm. This figure has been modified from 10. Please click here to view a larger version of this figure.
The ability of DNA gels to soften or stiffen before and after cell adhesion makes them an ideal model to study the role of dynamic tissue stiffness on cell function. All three designs have been used in mechanical and biological studies. However, all three designs have similar elasticities at various crosslink percentages, indicating crosslink length does not influence DNA gel elasticity (Table 2). In contrast, acrylamide concentration affects elasticity. These designs may differ in crosslinking kinetics since gel bursting tendencies, or the tendency of gels to dissolve in media or buffer, decreases with increasing crosslink length. However kinetic information on DNA gels is limited, but we do know an additional 30% of crosslinking occurs two days after L2 delivery to 50% DNA gels 11. Nevertheless, the generation of expansive and contractile forces from gel softening and stiffening, respectively, is unavoidable and cannot be decoupled 18,21. 100→70% gels generate about 0.5 Pa of stress 21, while 50→100% gels only generate about 0.04 Pa of stress since more energy is required to form bonds among the three stands of ssDNA 18. Therefore, the interpretation of results requires the consideration of stress and strain generated by the stiffening and softening of gels.
Technical challenges can arise when preparing DNA gels. The most frequent technical issue is gel bursting. Gel bursting can occur during several stages of DNA gel preparation including gel swelling, storage, and cell growth. Gel bursting is caused by multiple factors. First, gel bursting can be a result of improper solution composition. Common errors include insufficient dissolving of lyophilized ssDNA and improper pipetting of the viscous liquids. To avoid pipetting errors, raise the heating temperature to reduce gel viscosity for easier pipetting of solutions or aliquot the required amount of SA1 and SA2 polymerized solution prior to the second nitrogen bubbling step. Also, repeated heating of the polymerized solutions can partially evaporate the solution and increase solution viscosity. Adding a few microliters of TE buffer can help to reduce the viscosity. Second, gel bursting can be a result of poor DNA quality. QA/QC reports must be reviewed frequently. If QA/QC reports are acceptable and gel bursting still occurs, HPLC purified ssDNA can improve DNA quality. Third, gel bursting can result from insufficient DNA annealing. We have reduced the incidence of gel bursting by including annealing steps and rehybridization steps in the original protocol (see steps 1.3.6 and 2.8; 1.3.8 and 2.10, respectively). Gel bursting might be prevented by extending annealing and cooling times. However, additional exposure to heat can evaporate water to cause an increase in gel detachment from glass when gels are re-swelled. If detachment is excessive for any annealing time, the annealing steps can be eliminated (1.3.6 and 2.8).
Another frequent technical problem encountered is insufficient cell adhesion. The swollen gels are softer than the unswollen gels (Table 2) 18 making cell adhesion difficult. Furthermore, since each cell type responds differently to tissue stiffness, some cell types may experience this technical dilemma while other cell types do not. To resolve adhesion issues some of the follow steps should be considered: (1) use a high plating density, (2) alternate the ECM proteins conjugated to sulfo-SANPAH, (3) modulate the stiffness parameters, (4) increase ECM protein or sulfo-SANPAH concentration, and (5) increase incubation times. Residual toxic agents such as acrylamide monomer, APS, and TEMED could also contribute to lack of adhesion and cell death on gels. We have not experienced this issue since extensive washes incorporated into the protocol have proven sufficient, but we recommend performing Optional Step 3.2 if this issue occurs.
DNA gels can be utilized to more effectively study a variety of cell functions under dynamic or static conditions. We have shown that fibroblasts and neurons grown on DNA gels are affected by the modulation of gel elasticity. Since cell-matrix interactions are typically three-dimensional interfaces, information from three-dimensional dynamic matrix studies will help to produce more biologically relevant data. Therefore, we are currently translating this technology into a three-dimensional scaffold. We are developing a technique that replaces the polyacrylamide backbone with a more biocompatible material, while maintaining the DNA crosslinking technology. This new scaffold will serve as a three-dimensional, dynamic model and a tissue-engineering scaffold. Its biocompatibility will open opportunities for medical device applications.
The authors would like to thank: Dr. Frank Jiang, Dr. David Lin, Dr. Bernard Yurke and Dr. Uday Chippada for their contributions on developing the DNA gel technology; Dr. Norell Hadzimichalis, Smit Shah, Kimberly Peterman, Robert Arter for their comments and edits of this manuscript; funding sources including the New Jersey Commission on Spinal Cord Research (Grant #07A-019-SCR1, N.A.L.) and New Jersey Neuroscience Institute (M.L.P.); and publishers of Tissue Engineering, Part A for permission to reprint Figures 2 and 4 and Biomaterials for permission to reprint Figure 3.
idtdna.com Do not vortex ssDNA. Gentle invert the vial and/or pipette solution to mix.
PBS with calcium and magnesium Any brand.
10x Tris-Borate-EDTA buffer (TBE buffer) Sigma-Aldrich (St. Loius, MO) 93290 TBE is a reproductive toxin.
40% Acrylamide solution Fisher Scientific (Pittsburg, PA) BP14021 Acrylamide is a toxin.
Ammonium persulfate (APS) Sigma-Aldrich (St. Loius, MO) A3678 Prepare a 2% solution in TE buffer. APS is a toxin and irratant.
Tetramethylethylenediamine (TEMED) Sigma-Aldrich (St. Loius, MO) T9281 Prepare a 20% solution in TE buffer. TEMED is flammable, a corrosive, and a toxin.
24-well tissue culture plate Any brand.
proteochem.com or thermofisher.com C111 or 22589 Prepare a 0.315 mg/ml solution in water immediately before use. Dissolve at 37 °C and filter sterilze. It is normal to observe undisolved sulfo-SANPAH in the filter. Sulfo-SANPAH is light sensitive and, therefore, the solution should be protect from light until UV exposure.
Poly-D-Lysine (PDL) Sigma-Aldrich (St. Loius, MO) P6407 Prepare a 0.2 mg/ml solution in water and filter sterilize.
affymetrix.com 13813 Prepare a 0.2 mg/ml solution in 0.2 N acetic acid. Solution needs to remain cold at all times to avoid polymerization. Acetic acid is a flammable, toxic, and corrosive.
UV light source Place gels as close as possible to the UV light. UV light can cause skin or eye injury.
Balaban, N. Q., et al. Force and focal adhesion assembly A close relationship studied using elastic micropatterned substrates. Nat Cell Biol. 3, (5), 466-472 (2001).
Peppas Brannon-Peppas, L., Peppas, N. A. Dynamic and equilibrium swelling behaviour of ph-sensitive hydrogels containing 2-hydroxyethyl methacrylate. Biomaterials. 11, (9), 635-644 (1990).
Charati, M. B., Ifkovits, J. L., Burdick, J. A., Linhardt, J. G., Kiick, K. L. Hydrophilic elastomeric biomaterials based on resilin-like polypeptides. Soft Matter. 5, (18), 3412-3416 (2009).
Davis, K. A., Burke, K. A., Mather, P. T., Henderson, J. H. Dynamic cell behavior on shape memory polymer substrates. Biomaterials. 32, (9), 2285-2293 (2011).
Dembo, M., Wang, Y. L. Stresses at the cell-to-substrate interface during locomotion of fibroblasts. Biophys J. 76, (4), 2307-2316 (1999).
Gray, D. S., Tien, J., Chen, C. S. Repositioning of cells by mechanotaxis on surfaces with micropatterned youngs modulus. J Biomed Mater Res A. 66, (3), 605-614 (2003).
Homma, M., Seida, Y., Nakano, Y. Effect of ions on the dynamic behavior of an electrodriven ionic polymer hydrogel membrane. Journal of applied Polymer Science. 82, (1), 76-80 (2001).
Horkay, F., Tasaki, I., Basser, P. J. Osmotic swelling of polyacrylate hydrogels in physiological salt solutions. Biomacromolecules. 1, (1), 84-90 (2000).
Jiang, F. X., Yurke, B., Firestein, B. L., Langrana, N. A. Neurite outgrowth on a DNA crosslinked hydrogel with tunable stiffnesses. Ann Biomed Eng. 36, (9), 1565-1579 (2008).
Jiang, F. X., Yurke, B., Schloss, R. S., Firestein, B. L., Langrana, N. A. Effect of dynamic stiffness of the substrates on neurite outgrowth by using a DNA-crosslinked hydrogel. Tissue Engineering Part A. 16, (6), 1873-1889 (2010).
Jiang, F. X., Yurke, B., Schloss, R. S., Firestein, B. L., Langrana, N. A. The relationship between fibroblast growth and the dynamic stiffnesses of a DNA crosslinked hydrogel. Biomaterials. 31, (6), 1199-1212 (2010).
Kloxin, A. M., Tibbitt, M. W., Anseth, K. S. Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms. Nat Protoc. 5, (12), 1867-1887 (2010).
Lin, D. C., Yurke, B., Langrana, N. A. Mechanical properties of a reversible, DNA-crosslinked polyacrylamide hydrogel. J Biomech Eng. 126, (1), 104-110 (2004).
Lin, D. C., Yurke, B., Langrana, N. A. Use of rigid spherical inclusions in young's moduli determination: Application to DNA-crosslinked gels. J Biomech Eng. 127, (4), 571-579 (2005).
Luo, Y., Shoichet, M. S. Light-activated immobilization of biomolecules to agarose hydrogels for controlled cellular response. Biomacromolecules. 5, (6), 2315-2323 (2004).
Marklein, R. A., Burdick, J. A. Spatially controlled hydrogel mechanics to modulate stem cell interactions. Soft Matter. 6, (1), 136-143 (2010).
Pelham Jr, R. J., Wang, Y. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci U S A. 94, (25), 13661-13665 (1997).
Previtera, M. L., Chippada, U., Schloss, R. S., Yurke, B., Langrana, N. A. Mechanical properties of DNA-crosslinked polyacrylamide hydrogels with increasing crosslinker density. BioResearch Open Access. 1, (5), 256-259 (2012).
Previtera, M. L., Langhammer, C. G., Firestein, B. L. Effects of substrate stiffness and cell density on primary hippocampal cultures. J Biosci Bioeng. 110, (4), 459-470 (2010).
Previtera, M. L., Langhammer, C. G., Langrana, N. A., Firestein, B. L. Regulation of dendrite arborization by substrate stiffness is mediated by glutamate receptors. Ann Biomed Eng. 38, (12), 3733-3743 (2010).
Previtera, M. L., Trout, K. L., Verma, D., Chippada, U., Schloss, R. S., Langrana, N. A. Fibroblast morphology on dynamic softening of hydrogels. Ann Biomed Eng. 40, (5), 1061-1072 (2012).
Saxena, T., Gilbert, J., Stelzner, D., Hasenwinkel, J. Mechanical characterization of the injured spinal cord after lateral spinal hemisection injury in the rat. J Neurotrauma. 29, (9), 1747-1757 (2012).
Sundararaghavan, H. G., Monteiro, G. A., Firestein, B. L., Shreiber, D. I. Neurite growth in 3d collagen gels with gradients of mechanical properties. Biotechnol Bioeng. 102, (2), 632-643 (2009).
Wozniak, M. A., Chen, C. S. Mechanotransduction in development A growing role for contractility. Nat Rev Mol Cell Biol. 10, (1), 34-43 (2009).
Wuerfel, J., et al. Mr-elastography reveals degradation of tissue integrity in multiple sclerosis. Neuroimage. 49, (3), 2520-2525 (2012).
Zaari, N., Rajagopalan, P., Kim, S. K., Engler, A. J., Wong, J. Y. Photopolymerization in microfluidic gradient generators Microscale control of substrate compliance to manipulate cell response. Adv Mater. 16, (23-24), 2133-2137 (2004).


text:
Polygamist sects likened to mob
Federal task force, victim aid proposed
McClatchy Newspapers/July 25, 2008
By Dave Montgomery
Washington - A Senate committee on Thursday heard appeals for the creation of a federal task force to combat polygamist sects that Senate Majority Leader Harry Reid described as sophisticated organized crime rings.
Texas Atty. Gen. Greg Abbott was among those backing legislation sponsored by Reid (D-Nev.). The bill would establish a task force in the U.S. Department of Justice and assist victims of crimes committed by polygamist groups.
The hearing, which included testimony from two former sect members, spotlighted the Fundamentalist Church of Jesus Christ of Latter Day Saints (FLDS) led by Warren Jeffs, who was once on the FBI's Ten Most Wanted List. Jeffs and five unidentified followers were indicted Tuesday by a grand jury in Texas.
Reid told members of the Senate Judiciary Committee that polygamist sects are a "form of organized crime" that have spread into numerous states, as well as Canada and Mexico.
"I am not saying that they are the same thing as the crime syndicates that used to run Las Vegas," Reid said. "But they engage in an ongoing pattern of serious crimes that we ignore at our peril."
He cited a "web of criminal conduct that includes welfare fraud, tax evasion, massive corruption and strong-arm tactics."
Carolyn Jessop of West Jordan, Utah, who escaped an FLDS community in Arizona in 2003, said that Jeffs exerted a "tyrannical hold" on sect members and performed secret marriages between underage girls and older men. Boys and girls as young as 12 were forced out of public schools to work for FLDS construction companies and other businesses, in shifts that lasted from 6 a.m. until after dark, she said.
Dan Fischer, a former polygamist who now runs a foundation to help young people who have left the FLDS, said the sect has grown to "disturbing cult level proportions" under Jeffs' leadership.
"Without question, FLDS members will sacrifice self, family and children if directed to by their leader," he said.
Principle Voices, a polygamy advocacy organization, denounced Reid's bill. "If Reid truly cares about women and children in polygamy, then he should help them, not hurt them," the group said in a statement. "Principle Voices strenuously objects to any effort to characterize our families as anything but what they are: families."
The Mormon Church outlawed polygamy more than a century ago, but breakaway sects have continued the practice.
The FLDS was originally based at isolated locations in Utah and Arizona but has since moved to other states.
The indictments issued Tuesday charge Jeffs and four of his followers with sexually assaulting girls under the age of 17. A sixth member was indicted on three counts of failure to report child abuse.
FLDS representatives have accused the state of religious persecution.

redpajama_set_name: RedPajamaCommonCrawl


An individual should take their time when they are choosing their career because they will be doing it for the entire of their life. It is important for one to have passion of the career that they will choose so that they can be able to deliver the best they can when they are doing their activities. The mechanic is an expert when it comes to repairing the cars and they can do it within a short period of time. The mechanic can be able to do brake repair so that the people can be safe when they are driving their vehicles and they will be able to control their speed. It is important for the mechanic to ensure that they have all the necessary tools which will help them to complete their tasks which they need to give to their clients. The tools should always be made using the new technology so that they can perform some of the tasks faster than they were doing before they were upgraded.
It is important for one to look for a skilled mechanic who will repair their vehicle regularly and they will be sure that their vehicle is going to last for a long period of time without any problems. The mechanics have the ability to diagnose and know the kind of problem that the cars will be having and they will rectify it. A good mechanic is also required to have good communication skills which will assist them to build a good relationship with their clients and the clients will always get the car repair services from them. It is important for them to understand the clients so that they can be able to help them.
The mechanic should also introduce the new technology in their sector and use it to gain more income as they continue delivering their services to their clients. A mechanic should look for tools that they can use in their sector which have been made using the new technology and they are going to get some benefits of the new technology. It is important for the people to be up to date with what is happening in the technology world so that they can be able to implement it. The mechanics should offer advice to their clients so that they can always know what they ae supposed to do so that their cars can last for long without any problems. The clients must always follow the advice which will be given to them by the experts and they will have their car stay for long without any mechanical problem.


text:
Home Military US Marine veteran Grady Kurpasi is third American to go missing in Ukraine this week
US Marine veteran Grady Kurpasi is third American to go missing in Ukraine this week
Jane Nam
Former U.S. Marine Corps officer Grady Kurpasi, 49, who has not been heard from since April 23, is the third American to be reported missing in Ukraine this week.
Kurpasi arrived in Ukraine on March 7 after deciding to volunteer as a soldier in the Ukrainian war. Having served in the U.S. Marine Corps for 20 years, before retiring in November 2021, he felt that his skillset would be valuable to the cause.
The other two American soldiers to be reported missing include Andy Huynh, 27, and Alexander Drueke, 39, both from Alabama and both of whom have military experience.
Videos of Huynh and Drueke surfaced on Friday on RT, a Russian state-controlled international television network, showing they had been captured by Russian forces.
When asked to comment about the captures of American soldier volunteers, Biden reiterated on Friday, "I'll say it again, Americans should not be going to Ukraine."
Former U.S. Marine Corps officer Grady Kurpasi, who has not been heard from since April 23, is the third American to be reported missing in Ukraine this week.
State Department spokesperson Ned Price announced during a press conference on Thursday that a third American had gone missing, although the name was not said.
Kurpasi's wife confirmed to CNN, however, that it was her 49-year-old husband who had been identified as missing.
Kurpasi arrived in Ukraine on March 7 after deciding to volunteer as a soldier in the Ukrainian war. Having served in the U.S. Marine Corps for 20 years before retiring in November 2021, he felt that his skillset would be valuable to the cause.
He arrived in the country's capital of Kyiv by March 21, and close friends reported last hearing from him April 23 and 24.
According to a family friend, Kurpasi started "receiving small arms fire" on April 26 while stationed at an observation post, suggesting they were in crossfires with the Russian army.
The last known communication with the veteran was when he had radioed the Ukrainian military to begin firing back.
The other two American soldiers reported missing include Andy Huynh, 27, and Alexander Drueke, 39, both from Alabama and both of whom have military experience.
Unlike Kurpasi, videos of Huynh and Drueke surfaced on Friday on RT, a Russian state-controlled international television network, showing they had been captured by Russian forces. RT broadcast that the two were being held by Russian-backed separatist forces in the Donbass region of eastern Ukraine.
Despite the protections for prisoners of war under the Geneva Conventions, including the guarantee of humane treatment and fair trials, the Russian military has responded that foreign fighters, including the captured Americans, are considered mercenaries and are not protected.
Featured Image via U.S. Dept. of Defense
IOC plans for Russian, Belarusian athletes to compete under neutral flag at Asian Games
'I will not be silent': Biden calls for end to hate in White House's first Lunar New Year celebration
Elaine Chao fires back at Trump in rare rebuke after another racist attack
Aruna Miller becomes first Asian American elected statewide in Maryland


On July 31st, Teferi Melesse Desta, Ethiopian Consul-General in Guangzhou, visited GPHL. GPHL's Chairman Li Chuyuan and Deputy General Manager Zhang Chunbo met the delegation.
On July 31st, Teferi Melesse Desta, Ethiopian Consul-General in Guangzhou, visited GPHL, a leading Guangdong-based pharmaceutical enterprise. GPHL's Chairman Li Chuyuan and Deputy General Manager Zhang Chunbo met the delegation.
Li Chuyuan briefed the delegation on GPHL's recent development and said its total sales revenue had exceeded 100 billion yuan in 2017.
GPHL will continue to develop its four main business arms: pharmaceuticals, healthcare, marketing, and medical treatment, boosting three new business models, that is, e-commerce, capital finance and medical equipment, aiming to establish itself as a world-class company.
At present, Wanglaoji, GPHL's flagship product, has entered more than 60 overseas markets. "We see a huge pharmaceutical market in Ethiopia, and we are looking forward to more cooperation with the country," said Li.
Teferi Melesse Desta said he is aiming to promote the investment environment and opportunities in Ethiopia during this visit and welcomes GPHL to invest in the country's pharmaceutical industry. "There is huge potential to strengthen the partnership between China and Ethiopia. And I hope we can push forward the development of medical industry based on our unique market demands,"he said.
Located in East Africa, Ethiopia is one of the oldest independent countries in both Africa and the rest of the world. It is also located along the "Belt and Road", being a key distribution hub for surrounding countries. In recent years, the exchanges between China and Ethiopia have been increasingly frequent in terms of business and tourism.
In addition, Ethiopia is the first African country to emphasize the development of the pharmaceutical industry as one of its national strategies. Ethiopia has an enormous demand for medical treatment and medicines, but its local companies can satisfy only 20 percent of the market demand. Therefore, the Ethiopian pharmaceutical market relies heavily on imports, suggesting strong possibilities for international cooperation.
The visit of the Ethiopian CG in Guangzhou has deepened the mutual understanding between Ethiopia and GPHL and laid a solid foundation for further communication and cooperation.


Nothing shouts adventure like a Sailing Ship on the open sea. The classic mast and rigging hold back the power of the unfurled sails, driving the stately vessel through the waves. You get to write the captain's log on the fine linen pages of our luxurious faux leather refillable journal.
Perfect gift - no issues whatsoever.
Classy, durable, "special" journal that our son loves. He's a ship guuy anyway, but anyone who loves to write will really enjoy this product. Highly recommended.
This journal was exactly like I wanted. I use it for both business and personal notes each day..
I bought this as a gift. The cover was very nice and overall I was happy about the purchase. My only issue was the binding / paper which I found a bit cheep. However, I'm a paper snob and my friend really liked it.


text:
Ha rappresentato l'Australia all'Eurovision Song Contest 2022 con il brano Not the Same.

Biografia
Nato in Australia da padre filippino e da madre australiana, Sheldon Riley è salito alla ribalta nel 2018 con la sua partecipazione alla settima edizione della versione locale del talent show The Voice, dove si è piazzato 3º nella finale. Dallo stesso anno ha iniziato a pubblicare cover e brani inediti.

Nel novembre 2021 è stato confermato fra gli undici partecipanti di Eurovision - Australia Decides 2022, evento che ha selezionato il rappresentante dell'Australia all'Eurovision Song Contest, dove ha presentato l'inedito Not the Same. Nell'evento, svoltosi il 26 febbraio, il cantante si è piazzato al 2º posto sia nel voto della giuria che in quello del pubblico, ottenendo però abbastanza punti da venire proclamato vincitore e quindi rappresentante nazionale all'Eurovision a Torino. Nel maggio successivo, dopo essersi qualificato dalla seconda semifinale, Sheldon Riley si è esibito nella finale eurovisiva, dove si è piazzato al 15º posto su 25 partecipanti con 125 punti totalizzati.

Vita privata
Ha reso pubblica la sua condizione di persona autistica ed è apertamente gay.

Discografia

Singoli
 2018 – Fire
 2020 – More Than I
 2021 – Left Broken
 2021 – Again
 2022 – Not the Same

Note

Altri progetti

Collegamenti esterni

Partecipanti all'Eurovision Song Contest 2022
Persone che hanno fatto coming out

redpajama_set_name: RedPajamaWikipedia


text:
Early Political, Religious, and Other Americana from David Lesser Antiquarian Books
By Michael Stillman
David M. Lesser Fine Antiquarian Books latest catalogue, number 84, fits the mold of the typical Lesser catalogue. It is filled with rare books and ephemera of Americana, mostly from the 18th and 19th centuries, and each piece comes with its own story. Reading through a Lesser catalogue is reading through a series of vignettes of American history. By the time you are finished, you will know a bit more about the nation's past, and in particular, many of the small incidents that led to the major confrontations which shaped its history. Lesser's catalogues are not just book catalogues, but mini-history texts as well. Some of what we found follows.
Item 3 is more than a bit ironic as it is an Extract of a Letter from the House of Representatives of the Masschusetts-Bay proclaiming their loyalty to the British crown, even as it protests mistreatment in the days before the Revolution. The year was 1770, and the colony was upset with taxes, restrictions on imports, and heavy-handed British control of their institutions. Nevertheless, the representatives proclaim, "we cannot be justly suspected of the most distant thoughts of an independence on Great-Britain." For emphasis, they go on to say "it is so far from the truth, that we apprehend the colonies would refuse it [independence] if offered them; and would deem it the greatest misfortune to be obliged to accept it." Methinks they protested too much. They would have jumped on such an offer, but one was never made. Priced at $1,500.
Here is a different take on the British taxes that were a primary cause of the American Revolution. Rev. John Fletcher makes his arguments in American Patriotism Farther Confronted with Reason, Scripture, and the Constitution...with a Scriptural Plea for the Revolted Colonies. In this 1776 book, Rev. Fletcher defends the right of the British to tax the colonists. However, he goes a bit farther than most by claiming that paying British taxes is some sort of religious obligation. "The duty of paying taxes to the protective power, is so strongly connected with Christianity, that the Colonists must practically give up the scriptures, or submit to the reasonable demands of the British legislature," claims Fletcher. No wonder the founding fathers decided to separate church from state. Item 58. $1,500.
One of the uglier political papers of early America was James Callender's The American Annual Register...for the Year 1796, published early the following year. Callender was a shrill opponent of the Federalists, not above making use of scandal to achieve his goals. In this publication, he raises charges of financial dishonesty on the part of Alexander Hamilton relating to payments he made to James Reynolds. This forced Hamilton to reveal the truth - that he was paying Reynolds blackmail to keep quiet about the affair he, Hamilton, was having with Reynolds wife Maria. Item 21. $875.


Convert XLS, XLSX, and XLSM documents to PDF format.
Convert MS Excel files into PDF documents.
Convert Excel sheets to PDF files.
Convert your MS Word and Excel documents into PDF format.
Batch convert Excel 2003/2007 files to PDF accurately.
Batch-convert Excel spreadsheets into professional-quality PDF documents.
Convert PDF to EXCE, XLS, XLSX, and XLSM format.


text:
96 to be remade in Kannada
Trisha and Vijay Sethupathi's 96 become a solid hit in Tamil and has been loved by many. The film is also being remade in Telugu and Dil Raju is doing the honors.
Meanwhile, the latest we hear is that the same film will now be remade in Kannada as well. Star hero, Ganesh and Bhavana will be seen in the film which will be directed by Pritam Gabbi.
Meanwhile, the stars for the Telugu remake are still being casted by Dil Raju.


Beloved in Christ, how distressful and despairing can it be for one to realize at the end of his journey here on earth that he had been walking on the wrong side of the road? The Church teaches. Immediately after death the souls of those who die in a state of mortal sin descend into hell, where they suffer the punishments of hell's eternal fire. The chief punishment of hell is eternal separation from God, in whom alone man can possess the life and happiness for which he was created and for which he longs. The affirmations of sacred scripture and the teachings of the Church on the subject of hell are calls to the responsibility incumbent upon man to make use of his freedom in view of his eternal destiny. "Enter by the narrow gate; for the gate is wide and the way is easy, that leads to destruction.
"My Jesus, bind me to Yourself so that I may never more be separated from You. Make me entirely Yours before I die. I wish to love You ardently in this life, that I may love You ardently in the next. If in the past I have not loved You the way I should, now I turn to You with all my heart and soul. Give me, sweet Jesus, holy perseverance and grant me also the grace to ask for it.
Exercise: Reflect on the agonies of hell fire.


Take control of this high-value modern machine! Top features include front bucket seats, adjustable headrests in all seating positions, automatic temperature control, and power windows. It features an automatic transmission, front-wheel drive, and a refined 6 cylinder engine.


Like Italian food? Find the best deals on Italian Food in Hull with incredible vouchers.
This latest deal from Groupon is sure to delight anyone in Hull with a love of Italian food, vouchers for an Italian meal. Your pizzas will be so much tastier when served with savings of up to seventy per cent, and Hull's finest pasta dishes will taste extra tantalising with a side helping of fantastic discount vouchers. A voucher for Italian food in Hull is sure to please everyone, as nobody can deny the all encompassing allure of Italian delicacies, and everybody in Hull loves saving money with a discount voucher.
Immerse yourself in amazing Italian food and don't worry about the cost. Find amazing Italian food vouchers in Hull here.
The pleasantly rustic atmosphere of an Italian restaurant suits any occasion: birthdays, anniversaries, working lunches and romantic encounters - a perfect destination for dining out in Hull, which, thanks to this voucher, is now within the reaches of even the meagerest budget. Whether it is tiramisu or lasagne that whets your appetite for Italian food, these vouchers are indispensable for any and every keen diner in Hull. Savings like this are not to be sniffed at, so don't wait for a special occasion, get yourself a voucher and a few willing accomplices (they won't be hard to find), and head down to Hull tonight for a slap up Italian meal. Don't delay, because these vouchers are available for a limited time only, and you may find yourself paying full price for your next Italian meal in Hull.


text:
About OAB
EBT Medical Appoints Former J&J Executive to Board of Directors
Jeff B Smith joins EBT Board, bringing vast consumer healthcare experience.
Boston, Massachusetts and Toronto, Ontario – January 18, 2021 – EBT Medical, Inc. ("EBT") a venture-backed clinical-stage company reimagining the treatment for pelvic health disorders, is pleased to announce the appointment of Jeff B. Smith to the Board of Directors.
"I'm thrilled to join EBT as it rounds out its leadership, completes its first product, and races toward commercialization," said Mr. Smith. "The team has made tremendous progress to date, and given the compelling clinical data amassed, I see enormous potential to address multiple large markets, starting with overactive bladder. Especially at a time when patients are less inclined take another pill or undergo surgery, EBT offers the promise of treating patients in the comfort & safety of their own homes."
Jeff served in various key leadership roles at Johnson & Johnson, the world's largest and most diversified healthcare company. Most recently, he served as Company Group Chairman, North America and oversaw several divisions and brands including Tylenol®, Zyrtec®, Band-Aid Brand®, Neutrogena®, and others. During his time at J&J, he developed a strong track record of building and marketing global brands, driving insight-driven innovations, leading strategic business turnarounds, and delivering profitable growth.
Retiring from J&J, Jeff co-founded and is Managing Partner of Ignite Venture Studio, which develops brands to disrupt the 'startup-quo' by building and accelerating their performance to achieve success in collaboration with strategic partners. Recent launches include Paragon Vitamins, a subscription service personalized through metabolic testing, and Fluent Health, an integrated direct-to-patient solution for influenza and the common cold.
"I'm passionate about building brands that offer high-quality solutions to help people live their best lives every day. EBT is developing products that address major health issues in revolutionary ways, bringing neuromodulation into the mainstream," said Smith.
"We're delighted to welcome Jeff to our Board," said Keith Carlton, CEO of EBT Medical. "Jeff brings deep operational experience, an expansive network, and a very strong consumer healthcare background which perfectly complements our Executive Team and Board. We are thrilled to have his guidance and insights as we drive EBT forward."
About EBT Medical
EBT Medical was founded in 2014 based on scientific discoveries made at the University of Toronto with a mission to develop novel neuromodulation technologies that restore control, dignity and independence to those silently suffering from pelvic floor disorders. EBT's first product is a clinical-grade, noninvasive neurostimulator and associated ecosystem for overactive bladder (OAB). EBT has a significant patent estate around multiple neuromodulation technologies including issued patents on the Saphenous nerve for all pelvic health indications. EBT's management team and world-class advisory board have decades of relevant experience in pelvic health, neuromodulation, and consumer health. Learn more about EBT Medical at www.ebtmedical.com
About Overactive Bladder and Neuromodulation
Overactive bladder is a disabling syndrome affecting nearly 550M worldwide1. It is characterized by frequent, sudden, uncontrollable urges to void, often followed by incontinence episodes that are embarrassing and significantly impede activities of daily living. The major symptom of OAB, Urge Urinary Incontinence (UUI), is thought to be caused by over-signaling to & from the bladder and the pontine micturition center located in the brain stem. Over 50%2 of those with OAB remain undiagnosed and silently suffer, with marked increases in depression, anxiety, sleeplessness due to nocturia (waking to void), and social isolation. The economic impact of OAB in the United States was estimated at $76B including direct costs and indirect costs such as increased urinary tract infections, falls from running to the bathroom, and absenteeism3. Neuromodulation has been an established therapy for UUI since it was first approved in 1997.
Keith R Carlton
kcarlton@ebtmedical.com
1 546M affected individuals in 2018. Filipetto et al.: The patient perspective on overactive bladder: a mixed-methods needs assessment. BMC Family Practice 2014 15:96.
3$76B in 2015. J Manag Care Pharm. 2014 Feb;20(2):130-40. "Economic burden of urgency urinary incontinence in the United States: a systematic review."
Mike Labbe Joins EBT Medical as Vice President of Research & Development
Suranjan Roychowdhury, PhD Joins EBT Medical as Vice President of Clinical Affairs
EBT Secures USD $10M Series A
Ken Mariash Joins EBT Medical as Vice President of Marketing
EBT Wins $100K Cash Prize from MassChallenge at its Annual Awards
EBT Medical, Inc. Named a Top 26 Startup by MassChallenge Boston
© 2021 EBT MEDICAL
Reimagining the treatment of pelvic health disorders.
* Disclaimer: NiNA is currently in development and has not received market authorization for sale in the US, Canada, or any other geography.


text:
ASCO 2016 | Treatment options for stage III melanoma patients
James Larkin
James Larkin, MD, PhD, of the Royal Marsden NHS Foundation Trust, London, UK, explains the treatment options available for patients with stage III melanoma, which means that the disease has been cured with surgery but there is a risk of it coming back. According to Dr Larkin, treatment options are limited and usually participation in clinical trials is the recommended option for such patients. Last year, there was a trial comparing nivolumab and ipilimumab (IPI), and now there is a trial involving the use of pembrolizumab (PEMBRO) as a treatment option. For patients with stage III melanoma that cannot be removed using surgical means, there are several different treatment options (the use of drugs, for instance), but each patient is usually considered on an individualized basis. Recorded at the American Society of Oncology (ASCO) 2016 Annual Meeting held in Chicago, IL.
PEMBRO
Royal Marsden NHS Foundation Trust
Stage III melanoma
Ipilimumab, Pembrolizumab
More from James Larkin
ASCO 2016 melanoma highlights: immunotherapy developments and further data on checkpoint inhibitors
The importance of patient involvement in discussing treatment options
Availability of clinical trials for patients with brain metastases and more aggressive diseases


text:
01Cup
02Xfinity
03Gander Outdoors
01ARCA
02USAC/POWRi
03NKNPSE/NKNPSW
04Off-Road
USAC/POWRi
NKNPSE/NKNPSW
Antron Brown is Top Toyota at Topeka NHRA
Antron Brown advanced to the Top Fuel semifinals to lead Toyota at the Menards NHRA Heartland Nationals presented by Minties at the Heartland Motorsports Park in Topeka.
The three-time world champion has now advanced to the semifinals or beyond in three consecutive races and in four of the last six. With the strong run, he has climbed up to fourth in points, just nine points out of second.
In the opening round, Brown had the advantage at the starting line and never trailed in defeating Scott Palmer with a 3.740-second pass, his quickest of the weekend. He advanced to meet Doug Kalitta in an all-Toyota second round match-up after Kalitta had defeated Leah Pritchett in his opening run. Kalitta was able to make a solid pass, but Brown had the edge at the tree and led wire-to-wire with a 3.743 run.
For the third race in a row, the Matco Tools Toyota driver squared off with Steve Torrence, but Brown was up in smoke early, giving Torrence the win on a run that Brown would have defeated with his run in each of the first two rounds.
"We're definitely making gains, but we've been so close," said Brown of his semifinal run. "We're learning as we're going, though. Our Matco Tools Toyota dragster is running really, really good. I'm starting to get back on my game on the starting line, too. The more laps you get, the better you get. With that said, we just need to back it off a little more and you still run good ETs. We're looking good going into Bristol. We just need to keep doing what we're doing and maybe we can pop a win off there. I thought this was going to be the race where we got a win, but really I thought that over each of the last three races."
In addition to Brown and Kalitta each going rounds today, Richie Crampton also advanced to the second round with a victory over Terry McMillen before falling to Billy Torrence in round two.
In Funny Car, J.R. Todd earned a first-round bye after winning the pole position with a 3.924-second pass in qualifying. The number one qualifier was the 100th for Kalitta Motorsports. Todd then ran an uncontested 3.906 in round one. Unfortunately, he smoked the tires early in a second round loss to Robert Hight.
Hight had previously eliminated Shawn Langdon in the first round.
The Mello Yello Drag Racing Series is back in action next weekend at Bristol Dragway for the NHRA Thunder Valley Nationals, June 14-16.
Continue the Race
All Eyes Were on Toyota's Next Generation of Drivers
Kyle Busch Drives Camry to Runner-Up Finish in Kentucky
Bell Scores Runner-Up in Bluegrass State
They Said It
First-Time Win for Tyler Ankrum in the Truck Series
Toyota Drivers Achieve Top-10 Finishes Over Holiday Weekend
Legal Terms |
© 2019 Toyota Motor Sales, U.S.A., Inc.


← Honoring our Veterans-Thank you!
Today, Veterans Day, November 11, 2010, is the 40th anniversary of the POW/MIA bracelets.
"Over 1500 Americans are either Prisoners of War of Missing in Action in Southeast Asia.Aside from the inhumane treatment witnessed by those few who have returned, the most tragic aspect is that most of the families of these men do not know if their sons, husbands, or fathers are alive or dead. Hanoi won't tell them.
Those words were taken directly from a small card that came with the bracelets back in the 70s and 80s. I have two original bracelets that I recently found in a box of items my father had that had been packed away for years.
The two men whose names are on these bracelets have been accounted for. Sgt. Gary Pate MIA May 22, 1968, identified March 13, 2009, buried with crew/group in Arlington National Cemetery June 2010. I hope to visit his grave this December when I again volunteer to lay wreaths on the graves with Wreaths Across America. I'm sure this grave is in Section 60 (where my brother is buried) because that's where all the new burials are being placed. You can also find Sgt. Pate's name on the most recent "accounted for" list at the DPMO Office.
The other bracelet is for Cdr. Guy Johnson, MIA December 20, 1965. He was identified and laid to rest in 1977 also at Arlington National Cemetery. His story, his family story after his return is awesome. I don't know why these bracelets were not worn, as one of them is still in the original unopened package. I guess it's because dad was always wearing one with the name of his son [my brother] on it. I am going to locate the families and send them the bracelets.
My brother Capt. Herbert C. Crosby, US Army, MIA 1970, was a name on a bracelet for over 37 years. Through God's grace our prayers were answered and Herby was laid to rest in Arlington National Cemetery May 25, 2007. After the press release of his identification, I was contacted by many people who had a bracelet with his name on it. We've received almost 20 so far, and as recent as August 2010.
Displayed on his flag with other cherished items are four of the many we have received. My mother who is now in a nursing home wears one all the time that we received in August.
Each person has a story that goes along with the bracelet. One lady was cleaning her jewelry drawer out and found the bracelet. She wondered what happened to Capt. Crosby. The next day on the front page of her local (Idaho) newspaper was a photo of the horse and caisson carrying his flag covered coffin at his funeral and his story…this was on the very day she found the bracelet . She later personally returned the bracelet to my mother in Florida.
One of my brother's comrades was making a plaque to present to our family from the veteran's association of pilots he flew with in Vietnam (http://www.rattler-firebird.org/) need a bracelet for the plaque so did an internet search finding the office of National League of POW/MIAs in Ohio. He called asking if there was a way to find a bracelet with a specific name (needle in a hay stack). The State Coordinator asked who it was. He told her Capt. Crosby. She said, "he's on my arm!" She had worn his bracelet continuously since 1973. How ironic? Divine Appointment I'd say. This lady had also met my mother years ago at a POW/MIA meeting in Boston. She never took it off, even when going through airport security, except for once to have an MRI done. My brother she said had met some fantastic people over the years, and traveled over a good part of the globe. This is probably the most touching story because she wore that bracelet all the time.
A Vietnam veteran from Jacksonville, Florida, had a bracelet on his office desk all these years. When he found out the news he returned it, and also attended the funeral at Arlington National Cemetery (May 2007).
There are so many stories to tell but the fact is that people cared. They cared then and were brave enough to wear the bracelet, and they still care and still wear a bracelet.
When someone comes across their bracelet they'll do an Internet search and that's how they're finding me as I have a website dedicated to my brother at http://www.firebird91.org/. Also, there are now many veteran websites with the updated news that he is no longer MIA.
I have given most bracelets to family members who have asked to have one. My mother is wearing one of the more recent ones that came in this year.
Thank you everyone who wore, or is still wearing a POW/MIA bracelet. Never forget our fallen heroes.
Read here about the Origin of original POW bracelets. to learn how they came to be.
Dave Berner is a documentary producer. His most recent audio work is FINDING MY KEROUAC and PEBBLE BEACH STORIES. He is also the author of Accidental Lessons: A Memoir of a Rookie Teacher and a Life Renewed, along with hundreds of magazine articles and broadcast reports for CBS Radio Chicago, NPR, and ABC radio.. He teaches at Columbia College Chicago.
Dave interviewed me while putting together a documentary about the bracelets last year although his final piece was on one family's story. You can find more information at http://braceletsofgrace.blogspot.com/ His radio documentary about the POW/MIA bracelets of the Vietnam War era – BRACELETS OF GRACE will be aired on 11/11 on the Prairie Public Network. This is a large network of public radio stations, some 8-12 stations. They will be airing an edited version of the documentary at 3:30pm and 7:30pm central time tomorrow, November 11.
The broadcast can be listened to through station streaming at this site –http://www.prairiepublic.org/radio/listen-now/ If you have time listen in.
Happy Anniversary POW/MIA bracelets. I'm sorry you're still in need, yet I'm happy you are still around to remind us all that we shall NEVER FORGET our fallen, from all wars.
This entry was posted in POW-MIA Bracelets, POW/MIA, Salute, Stories and tagged MIA, pow, POW-MIA Bracelets, Vietnam. Bookmark the permalink.
It's still tough…I get misty-eyed just reading the stories, knowing, as you indicated, that there are just too many "coincidences" involved for there not to be some sort of supernatural guidance involved.As you know Mary Lou, I've been wearing a POW/MIA bracelet for over 38 years. The individual on mine was a hero named Capt. James Reed. If you are interested you can read his story here.My bracelet is made of Stainless Steel…A good thing, because it is still structurally sound even though Capt. Reed's name is beginning to be difficult to read after all these years of being rubbed against one surface or another. But I too made the promise to wear it until we know his status, and I intend to keep that promise.God Bless Capt. Reed, Capt. Herb Crosby, and all those selfless women and men, living and dead, who raise their right hand and swear to "Support and Defend", protecting us ALL from evil.


text:
The RCMP logo is seen outside Royal Canadian Mounted Police "E" Division Headquarters, in Surrey, B.C., on Friday April 13, 2018. A British Columbia man who is now in his late 70s has been found guilty of numerous counts of sexual assault.THE CANADIAN PRESS/Darryl Dyck
B.C. man guilty of sex assault after befriending victims at Metro Vancouver churches
Raymond Gaglardi offered therapy sessions to the victims after meeting their parents at the churches
A British Columbia man who is now in his late 70s has been found guilty of numerous counts of sexual assault.
Coquitlam resident Raymond Gaglardi appeared in B.C. Supreme Court in New Westminster on Monday, and court records show he was convicted on 11 counts.
The charges related to historical sexual assaults of young men or teenage boys who attended several Coquitlam-area churches between 1993 and 2007.
RCMP said in 2020 that the offences happened at Gaglardi's home, where he offered therapy sessions to the victims after meeting their parents at the Metro Vancouver churches.
An investigation began in 2019, and the first charges were laid the following year, when Gaglardi was 75 years old.
He is scheduled to return to court for a pre-sentence report on Dec. 8.
RELATED: VIDEO: How the Mormon church 'help line' hid child sex abuse
CrimeMetro Vancouversexual assault
Vote in Ukraine's Russia-held areas stokes tension with West
'Shocking' erosion of sand dunes in Prince Edward Island National Park due to Fiona


Flew into cloud at high speed at 1200hrs on 14 Jan 1944. Aircraft crashed at Rosenden about 4 miles from Wooler. Buried in Yorkshire. Son of Joseph and Maud A. Lookyan, of Port of Spain, Trinidad.
Additional information: Son of Joseph and Maud A. Lookyan, of Port of Spain, Trinidad.
Grave/Memorial Reference: Sec. C. Row J. Grave 4.
Look-Yan was killed in Hurricane Z3076 of 59 OTU which crashed at Roseden, 4m SE of Wooler, Northumberland.


Some brands just know how to get it right. Earlier in the year, we looked at a few brands with some pretty amazing content marketing strategies. From Red Bull to Whole Foods, there are plenty of companies that are driving the conversation and staking out a position for themselves by creating epic content.
Sure, content marketing is quickly becoming the norm; we're seeing an increasing number of companies devoting larger chunks of their marketing budget for content. Even still, it's not an easy task to execute—it requires creative and strategic planning, a newsroom mentality, solid writing and content production skills, and a change in one's marketing philosophy.
And since this is all easier said than done, let's give kudos to three more brands that have found content marketing success.
Although it's going to take some time before P&G develops content for all of its brands, they've done an excellent job so far. Let's start with the Pampers microsite. Aside from the obvious diaper promos, parents can also find a ton of helpful information that's all about taking care of baby. Featuring tips on sleep to nutrition to skin care, Pampers.com and all of its distribution channels, such as Twitter and YouTube, have become great resources for moms and dads.
P&G also runs sites geared towards specific consumer demographics. The colorful BeingGirl.com is designed to educate pre-teen/teenage girls about their changing bodies and other issues currently affecting them (beauty, fashion, relationships). And then there's the Martha Stewart-esque HomeMadeSimple.com, which offers interior design ideas, recipes, crafts, and more for the older consumer. By catering to a wide range of their consumers, they've become a go-to for their customers.
It's no surprise that a store that specializes in kitchen goods would have an appropriately themed content section, but their blog Taste is a step above the traditional foodie site that's perfect for fine-dining lovers. Get dinner party ideas, learn how to preserve strawberries, or discover how to grow your own vegetable garden. They drive awareness through a robust social media presence, engaging with consumers on a variety of platforms, including Pinterest, Instagram, and YouTube. Whether you're looking for recipe ideas or need to watch a chef prepare a dish step-by-step, you'll find just what you need.
That's right—content marketing isn't just for the fun-loving food and retail industries. It can work for a variety of brands too. Although OpenForum.com is a microsite that provides content geared towards entrepreneurs and small business owners, I'd argue there's something here for anyone in the professional world; the site's content ranges from tips on management and productivity skills to updates on Google's SEO algorithms to infographics on the changing CFO. And in doing so, AmEx becomes a company that doesn't just provide you with a credit card, but one that also helps you achieve your entrepreneurial goals. And that's a pretty cool thing.


text:
Q: How to Get Results From a Background Process I am designing a Ruby on Rails application that requests XML feeds, reads them in, and parses them into objects to be used in views. Since the request for the XML feed and subsequent receipt of it can take several seconds from some sources to complete I need a way to offload these tasks from my front-line application tier. I do not want my application servers to take more than a few hundred milliseconds to process a request. Currently the application serving processes sit and wait for the XML feed data to be returned so they can parse it and finish return the user's request. I am aware of DelayedJobs, however given that the result of this action is to be returned to the user in real-time I am unsure of how to offload it to a background task and receive the result.
If I offload this task to a background task how does the result get returned to the user loading the page?

A: One common model for this sort of thing is to use your preferred background job library (you mention DelayedJob, which seems to be a popular one) to offload the task from the request/response cycle, and then set up AJAX polling on the client to update the page with the results once they become available.

A: If you are open to Perl code running on your server, I'd lift a piece of LiveJournal infrastructure: Gearman and TheSchwartz
Sounds like you want Gearman - and it has Ruby client bindings.
(see
http://www.livejournal.com/doc/server/lj.install.workers_setup_install.html )

A: You can have your main returned page fire an AJAX request at a second tier of servers that handle the XML retrieval, and return HTML for the section of the page that will contain that information.  That way you aren't running any asynchronous jobs (from the server's point of view) and the retrieval won't start until the AJAX request comes in, which will reduce the bandwidth you waste on bots.
This is a standard use of AJAX, so I'm not sure whether I'm missing something in your problem that makes it inappropriate for you.

A: The most common approach is to use AJAX and DelayedJob here, but it is only an usability improvement - instead of user waiting for 5sec to load the page they get an empty or half-empty page with a spinner for 5 seconds. The only way (in my opinion) to really improve the user experience is to load and process those xml feeds periodically and display to user the cached result.

redpajama_set_name: RedPajamaStackExchange


text:
NEW HAMPSHIRE MONEY PARTY CANDIDATE KATRINA SWETT CAN'T GET THE LIEBERMAN STINK OFF
317 days ago Lieberman fake-Democrat Katrina Swett (co-chair of his pathetic 2004 presidential "campaign") was using typical DLC/Lieberman talking points to chastise progressives and grassroots Democrats for rejecting a treacherous corporate whore and warmonger like Lieberman. It wasn't even a year ago and Swett was still all sweaty with Liebermania:
Swett believes Lieberman lost because of three perceived Democratic "sins": the sin of supporting the Iraq war and being tough on defense, the sin of being bipartisan and the sin of displaying religious faith. Swett said those traits might make Lieberman undesirable to many Democrats but they could be key for Democrats in winning future national elections.
"Round two in Connecticut is going to be a battle between two Democrats: Joe Lieberman, a centrist Democrat, and Ned Lamont, a pretty-far-left-of-center Democrat," said Swett. "I'm convinced that Joe Lieberman is the better leader . . . and I'm also convinced that he's the better positioned politically for the future of the party that I love."
The party she loves? That would be the Money Party, represented on her side of the aisle by the corporately sponsored DLC. But today, in the hopes of advancing her floundering career and taking over the New Hampshire Senate seat of another Money Party shill, Republican John Sununu, Swett is singing a different tune, a very different tune. She can't get far enough away from Lieberman, nor fast enough. In her desperation to not be associated with the Lieberman branch of the party she has always been a part of, she is donating money to progressive Rep. Tom Allen, the progressive running against Lieberman's candidate in Maine, the hapless Republican rubber stamp Susan Collins. Swett not only donated $1,000 to Allen, she did it through MoveOn.org, a group she has chastised as being a bunch of left-wing extremists. Swett is the absolute worst kind of Democrat you'll ever find-- and the only way to ensure that John Sununu is re-elected.
Meanwhile, the race keeps heating up. Like Swett, Portsmouth Mayor Steve Marchand has been campaigning for months. Jay Buckey, a Dartmouth medical professor and former astronaut, threw his hat in the ring this month. Meanwhile, many Democrats are publicly pining for former governor Jeanne Shaheen, led by former Democratic party chairwoman Kathy Sullivan, who is leading a Draft Shaheen campaign.
I hope that by election day we will have a page up entitled: Vote No On Lieberman Treachery, with a list of all the candidates he is endorsing. Meanwhile, I want to urge you to contribute to Tom Allen, Donna Edwards and Victoria Wulsin and to withhold donations from Katrina Swett.
Labels: Blue America, Katrina Swett, Lieberman, New Hampshire
posted by DownWithTyranny @ 12:22 PM
At 12:32 PM, fahrender said...
right on, baby. show katrina up for the devious shill she was born to be .......
IMMIGRATION BILL CLEARS A PROCEDURAL HURDLE THROWN...
WILL DOOLITTLE GET A HARSHER SENTENCE THAN CUNNING...
WILL THERE BE A MUD-WRESTLING MATCH BETWEEN RICHAR...
WHO TO BLAME FOR TODAY'S SERIES OF DISASTERS AT TH...
A BETTER IDEA FOR A MEREDITH VIERA INTERVIEW
NOW WHY DO MORMONS THINK THEY'RE TOO GOOD FOR THE ...
WILL JOE LIEBERMAN ENDORSE AL WYNN? STEVE BLACK? ....
EVEN THE WORST BUCKET OF SLIME GETS IT RIGHT SOMET...
DEMOCRATS COULD TAKE VIRGINIA AND TENNESSEE SENATE...
IS McCAIN'S STRAIGHT-TALKER BULLSHIT GOING TO BACK...


[HT DownloadSquad] DepthJS from Fluid Interfaces on Vimeo. We are long overdue for a change in the way we interact with our machines, I hope this is more than a passing fad.
[HT Fat Angus] Here's the Stanford page for more info.
Do the images you post online pose a security risk?


Is It Important to Hire Lawn Care Services?
It is common for a house owner to want to have a well maintained and well trimmed lawn. When it comes to doing your own lawn care, you should understand that it is a lot easier said than done. But do not worry too much about this because you can actually hire lawn care services to help you. If you hire a lawn care service, then you are sure to receive quite a lot of great benefits. This article is for anyone that wants to know and understand some of the best benefits to lawn care services. Here, you will learn about some of the best benefits that lawn care services can provide for you. So without further ado, here are the top 3 benefits.
For one thing, lawn care services provides great knowledge and experience; and this is the first great benefit. The reason why knowledge is needed is because lawn care services take skill and understanding. The reason why experience is needed is so that the lawn care services can use their knowledge in the best way possible. You will really be able to have a great and well maintained lawn because of this knowledge and experience that lawn care services provide. Knowledge and experience is the first benefit that you will receive from lawn care services.
For another thing, when you hire lawn care services, you will be able to experience true convenience. When you do your own lawn care, you can probably think of a lot of inconveniences that you will have to face. The inconveniences will become even greater when you are always on a tight schedule. But you can be sure that when you hire lawn care Okemos services, you will experience true convenience because they will take the full responsibility out of your hands and into their own. This is benefit number two to hiring lawn care services.
Providing their own tools and equipment is yet another great benefit to lawn care lansing services. This is a great benefit for two reasons. Firstly, this benefit ensures that you no longer have to provide them the tools and equipment needed to maintain and trim your lawn. For another reason, you can be sure that the tools and equipment they bring are some of the best and highest in quality; ensuring that your lawn is maintained in the best way possible. These were the two reasons why providing their own tools and equipment is such a great benefit. Just because this was the last benefit that we mentioned here in no way means it is the least of the benefits.


text:
The Heinrich Böll Foundation is committed to a critical dialogue on climate and resource policy in Brazil. It also promotes debate and analysis on security and gender policies, the right to the city, the influence of religion in politics and society, and the crisis of political institutions.
It supports national and international exchange on the topics we work on and the participation of civil society organizations and networks in the political dialogue between science, civil society and government institutions. Classical human rights work is still being promoted.
In 2014, Brazil's rapid economic growth, which marked the first decade of the century, came to an end. The economic crisis was the political crisis. Triggered by numerous corruption scandals, it led to the impeachment of former President Dilma Rousseff (PT) in mid-2016 and ends today, 2017, as a massive loss of credibility for the political elites of all parties. In the new political framework, organized civil society must also explore new possibilities of influence. Here, the foundation offers space for debate and promotes dialogue and networking.
The government is trying to boost economic development with radical reforms (labor law, pensions) and the privatization of public infrastructure. Large-scale projects are planned and, in some cases, already started, among others with China, for the logistical development of the Amazon and its resources. In the sensitive ecosystem of Cerrado, in northeastern Brazil, a new, huge soybean cultivation area is to be created with government support. Environmental and social aspects are given little consideration. People who work to protect resources and the environment are increasingly threatened. Here, the Heinrich Böll Foundation supports the partner organizations FASE Amazônia and SDDH as well as the development of networks that visualize the effects of these projects and developments on people and the environment and try to influence political decisions.
Brazil is internationally regarded as a test country for instruments of the Green Economy, in particular for compensation mechanisms in the climate, energy and resource policy. New legislation anchors payments for environmental services (PES), such as REDD + and TEEB, which consider land, forests, but also biodiversity as resources, CO2 storage and investment objects. For many years, the Brazil Office and its partners (Grupo Carta do Belém, FASE, Terra de Direitos, INESC) have been critically examining the implications and alternatives to the new policy instruments. As one of the largest agricultural producers in the world, Brazil is steadily expanding its acreage for agricultural products. Nowhere in the agricultural sector per capita comparison are more harmful pesticides used than in Brazil. To preserve its unique biodiversity, it is becoming more and more important to take legal action against the privatization of seeds, the loss of biodiversity and the use of pesticides, as our partner Terra de Direitos does.
After the organization of major sporting events (World Cup and Olympic Games), the state of Rio de Janeiro is bankrupt. The security situation is more dramatic than it has been for many years, also because many social and prevention programs have been discontinued. As the police and military encroachment on the poorer population has increased sharply, the partner organization Justiça Global is again more concerned with direct human rights work, but also tries to influence the design of security policy. Here, too, the foundation supports networking. 2017 is clear that the games have left little positive legacy. The inhabitants of the slums therefore continue to organize themselves to point out structural problems in Rio de Janeiro and demand changes. Here the foundation works a.o. with the Instituto Raizes em Movimento and the Observatorio das Favelas in the preparation of studies on the key topic of mobility together. Partner in this context is also the research center BRICS Policy Center.
In the fight for women's rights and against homophobia, the growing influence of religious - evangelical and conservative-Catholic groups on society and politics has given way. The influence of women's organizations on the work of thematic advisory councils, which are directly involved in many political decision-making structures in Brazil, has declined significantly. Here the Heinrich Böll Foundation supports the inventory of these regression steps and the reflection on new possibilities of influence of the women's movement, i.a. together with the women's rights organizations CFEMEA, SOS Corpo and SOF. Together with the Institute for Religious Studies ISER, the foundation offers publications and events on the area of conflict between religion and politics.
In view of the loss of credibility of politics, the Heinrich Böll Foundation will increasingly focus on the growing spheres of influence of companies, churches, and others. to discuss political decisions in Brazil and Latin America.
Büro Rio de Janeiro, Brasilien
Director: Annette von Schönfeld
Fundação Heinrich Böll
Rua da Glória 190, Ap. 701
2011180 Rio de Janeiro – Gloria
Fon: +55-21-3221 9900
E-Mail: br-info@br.boell.org
Website: www.br.boell.org
Twitter: www.twitter.com/hbsbrasil
Facebook: www.facebook.com/bollbrasil
Instagram: www.instagram/heinrichbollbrasil


What arawards did James Patterson win?
What common characteristics do linear and nonlinear equations have? Who won the baseball Organization of the Year Award in 2005? Who won the Organization of th Year Award in 1983? When was James Patterson Lyke born? Who won the Organization of th Year Award in 1982?