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Griffin:shRNA Transfection
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shRNA Transfer Vector Overview
Short hairpin RNAs (shRNAs) are typically modeled after miRNA hairpin precursors and cloned into a transfer vector that is suitable for either direct transient transfection of the shRNA, or for packaging into a lentiviral particle. Lentiviral transfer vectors are ~8-10 kb in length.
Transient shRNA Transfection
Instead of chemically synthesizing the siRNAs before introducing it in the cell, the siRNAs are made directly by the cells through an expression vector that is transiently transfected into a dividng cell. The shRNA transfer vector alone can be transiently introduced into the dividing cell where the shRNA is synthesized by cellular machinery. While transient transfection is advantageous for fast analysis of shRNA mediated effects, stable transfection ensures long-term, reproducible as well as defined shRNA effects.
Stable shRNA Transfection
For many disease models, the most desirable cell types such as immune system or primary cells are not amenable to transfection. Viral delivery of RNAi vectors is a powerful alternative to transfection for these cell types as well as for in vivo applications. Stable expression is achieved by integration of the gene of interest into the target cell's chromosome: Initially the shRNA of interest has to be introduced into the cell, subsequently into the nucleus, and finally it has to be integrated into chromosomal DNA.
Stable expression can be influenced by two factors: The transfection method used and the vector containing the shRNA of interest. The transfection method determines which cell type can be targeted for stable integration through antibiotic selection. While many lipofection reagents transfect DNA up to a certain amount into adherent cell lines, efficient delivery of DNA into difficult-to-transfect suspension cell lines or even primary cells is only possible with viral methods and nucleofection. Nucleofection is a non-viral method of introducing DNA molecules efficiently into the nucleus of dividing cells, therefore significantly increasing the chances of chromosomal integration of the transgene. The technology was pioneered by Amaxa
Vector dependent
Although there is still some debate as to the effectiveness of this approach, a regular shRNA transfer vector may be able to integrate into the genome of the target cell by antibiotic selection alone. The process may occur randomly by the cell's machinery itself, possibly via DNA repair and recombination enzymes. If this phenomenon does occur, integration into inactive heterochromatin may result in little or no shRNA expression, whereas integration into active euchromatin may allow for shRNA expression. However, random integration could also lead to silencing of the shRNA cassette. Several strategies have been developed to overcome the negative position effects of random integration: Site-specific, homologous and transposon-mediated integration strategies are used but require the expression of integration enzymes or additional sequences on the plasmid.
Lentiviral particle dependent
Lentiviral particles are highly efficient at infection and stable integration of the shRNA into a cell system. To obtain the lentiviral particle, the transfer vector that contains the shRNA cassette is already flanked by LTRs and the Psi-sequence of HIV. The LTRs are necessary to integrate the shRNA cassette into the genome of the target cell, just as the LTRs in HIV integrate the dsDNA copy of the virus into its host chromosome. The Psi-sequence acts as a signal sequence and is necessary for packaging RNA with the shRNA into pseudovirus particles. Viral proteins which make virus shells are provided in the packaging cell line (HEK 293T), but are not in context of the LTRs and Psi-sequences and so are not packaged into virions. Thus, virus particles are produced that are replication deficient. Lentiviral particles can infect both dividing and nondividing cells because their preintegration complex (virus "shell") can get through the intact membrane of the nucleus of the target cell.
- Lentiviral systems efficiently transduce both dividing and non-dividing cells
- Study long-term gene knockdown with stable expression
- Reproducibly transduce cell populations
- Inducible or constitutive gene knockdown
Lentiviral Particle Overview
Lentiviral vectors are usually created in a transient transfection system in which a cell line (HEK293T) is co-transfected with three separate plasmid expression systems. These include the transfer vector plasmid (portions of the HIV provirus & shRNA cassette), the packaging plasmid or construct, and a plasmid with the heterologous envelop gene (ENV) of a different virus. The three plasmid components of the vector are put into a packaging cell which is then inserted into the HIV shell. Pseudoviral particles are then collected and must undergo titration to measure infectivity prior to experimental use.
Transfer Vector Plasmid
The transfer vector plasmid (~8-10 kb) contains cis-acting genetic sequences necessary for the vector to infect the target cell and for transfer of the shRNA cassette, and contains restriction sites for insertion of the shRNA. The 3' and 5' LTRs, the original envelop proteins, and gag sequence promoter have been removed. The shRNA transfer vector alone can be transiently introduced into the dividing cell where the shRNA is synthesized by cellular machinery.
In order to culture the transfer vector effectively and avoid recombination events, a suitable e. coli strain Invitrogen MAX Efficiency® Stbl2™ Competent Cells SKU# 10268-019, and culture temperature; 30°C expression, incubation temperature is important to optimize performance of the competent cells to produce intact lentivector.
However, there are distinct differences between E. Coli-dependent vector-based recombination events versus point mutations. Both can influence the fidelity of the lentivector, however the implications of point mutations in the construct is much more significant. Since the labor costs associated with cloning and production of the shRNA constructs are significant, be sure to invest your time in a transfer vector that is proven in literature or contains lot specific data for adequate controls from the vendor. DO NOT assume the vector you are purchasing on paper is the one you receive in the vial.
lentivector pSD31 for constitutive shRNA expression and pSD400 for conditional shRNA expression
Constitutive transfer vectors
Currently, most siRNA expression vectors are engineered to drive siRNA transcription from the polymerase III promoters U6 and H1. These promoters are particularly suited for hairpin siRNA (shRNA) expression, since they contain all of the cis-acting promoter elements upstream of the transcription initiation site and deploy a polyT transcription termination site that leads to the addition of 2-nucleotide (nt) overhangs (UU) to shRNAs, a feature that is important for siRNA function.
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Inducible (conditional) transfer vectors
Tetracycline Operator
Specific inhibition of gene expression using a stably integrated siRNA (Tiscornia et al. 2003) is not suitable for silencing genes that are involved in cell growth or survival (Zhou et al. 2006). In such cases, regulating siRNA expression in mammalian cells has become essential. Having the ability to control when and how much of a particular siRNA is expressed makes it possible to study temporal- and concentration-dependent effects on a host system and allow cells to grow prior to gene silencing by "toxic" siRNAs. Furthermore, studying a host cell's response to the presence and absence of siRNA often leads to further understanding of the target gene's function.
The inducible RNAi system uses a modified form of the regulated, tetracycline-controlled gene expression system described by Gossen and Bujard (2002) in mammalian cells for the purpose of temporal silencing target genes. This system relies on two components: a tetracycline regulatory protein (TetR), which has affinity to the tetracycline operator (TetO); and a TetO-tethered pol III promoter, whose transcription activity is blocked by binding to the regulatory TetR. In the absence of tetracycline (Tet), the TetR protein binds to the TetO sequence within the promoter and acts as a potent transcription suppressor. The shRNA expression is restored when the cell culture medium is added with either Tet or doxycycline (Dox), a Tet derivative, which binds to and causes dissociation of TetR from the pol III promoter via a TetR conformation change (Zhou et al. 2006). This system allows for observation of the loss-of-function phenotypes under noninduced and induced conditions in the same isogenic cells, excluding other potential interferences.
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Envelope Gene Plasmid
The third plasmid's envelope gene of a different virus specifies what type of cell to target and infect. Normally HIV can infect only helper T-cells because they use their gp120 protein to bind to the CD4 receptor. However, it is possible to genetically exchange the CD4 receptor-binding protein for another envelope protein that codes for different cell types. This gives the HIV lentiviral vector a broad range of possible target cells. There a few types of heterologous envelope proteins.
There are two major points to consider when choosing which coat (pseudotype). First, because VSV-G pseudotyped virus may infect human tissue, it may not be preferable to use if one is working strictly in a mouse system. Working with human infectious agents will likely require a heightened BLS lab safety level. A second point of consideration is the limitations imposed by the ecotropic coat: it is difficult to concentrate the virus due to the inherent instability of the ecotropic coat. Virus ultracentrifugation concentration is often necessary when infecting certain cell types or when generating transgenic mice, and for this reason, many choose to use VSV-G pseudotyped virus because the particles are stable to the high g-forces of ultracentrifugation. Other Polyethylene Glycol based concentration methods exist that may effectively address this concern. Even with highly concentrated virus, there is a possibility that the VSV-G or ecotropic pseudotypes may still display differing cell-type specificities for infection, an important consideration when planning an experiment. For example, in the CNS, VSV-G mediates vector entry primarily in neurons, while astrocytes appear to be better infected using other envelopes.
There are a growing number of examples of viral coat proteins for pseudotyping the virus that influences the tropism of infection in cell type in vitro, and tissue/organ type in vivo. For in vitro use, envelope glycoproteins and culture conditions employed should be carefully evaluated in order to address variable infectivity and phenotype awareness for each application.
Envelope (viral coat) proteins
Ebola virus
(Filovirus): Allows transduction of apical surface airway epithelium. Zaire subtype EBO virus glycoproteins (GP) EboZ pseudotypes were able to efficiently transduce after apical airway epithelial application. Broad tropism for mammalian cell types.
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Lymphocytic choriomeningitis virus (LCMV)
A noncytopathic arenavirus shown to infect a broad range of different cell types. Less inflammatory with tropism for the liver.
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Marburg virus
(Filovirus): Allows transduction of apical surface airway epithelium. Broad tropism for mammalian cell types.
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Mokola lyssaviruses (MK-G)
A neurotropic virus causing rabies disease. Targets neuronal cells and neural stem cells.
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Rabies virus
Target neuronal cells and neural stem cells.
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A chimeric envelope glycoprotein made of the extracellular and transmembrane domains of the feline leukemia virus RD114 and the cytoplasmic tail of the murine leukemia virus amphotropic envelope. Transduces primary lymphocytes; RD114/TR-pseudotyped vectors showed augmented transduction of human and macaque primary blood lymphocytes and CD34+ cells.
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Ross River virus (RRV; alphavirus)
Less inflammatory with tropism for the liver. RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype.
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Semliki Forest virus (SFV; alphavirus)
VSV-G and RRV pseudotypes have comparable physical titers, but infectious titers with the RRV pseudotype are lower than with VSV-G. Incorporation of SFV glycoproteins into lentivirus vector is less efficient, leading to decreased physical and infectious titers. The transduction rates with VSV-G-, RRV-, and SFV-pseudotyped lentivirus vectors into adherent cell lines can be significantly increased by using a combination of Polybrene and plates coated with CH-296 recombinant fibronectin fragments.
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Sindbis virus (SINV; alphavirus)
Sindbis virus encodes two transmembrane envelope proteins, E1 and E2. E2 is responsible for receptor binding; E1 is responsible for pH-dependent fusion. SINV when modified to contain the Fc-binding domain of protein A, this envelope gives a significant enhancement in specificity in combination with antibodies specific for HLA and CD4 relative to that without antibody.
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Venezuelan equine encephalitis virus (VEEV; alphavirus)
Venezuelan equine encephalitis virus (VEEV) is a distant relative of SINV, RRV, and SFV and is a representative of the New World alphaviruses. It is an arbovirus that is normally maintained in a "silent" enzootic cycle between mosquitoes of Culex (Melanoconion) spp. and rodent reservoir hosts.
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Vesicular stomatitis virus G glycoprotein (VSV-G)
VSV is broadly tropic and highly efficient at particle production. Broad tropism. Less inflammatory with in vivo tropism for the liver. Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. Two important parameters of the VSV-G-pseudotyping phenomenon relating to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions.
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Packaging Plasmid
The packaging plasmid is the backbone of the virus system. In this plasmid are found the elements required for vector packaging such as structural proteins, HIV genes (except the gene env which codes for infection of T cells, or the vector would only be able to infect these cells), and the enzymes that generate vector particles.
Most HIV lentivector systems require only 3 HIV encoded proteins, Gag, Pol, and Rev.
The RNA genome of HIV consists of at least 7 structural landmarks (LTR, TAR, RRE, PE, SLIP, CRS, INS) and nine genes (gag, pol, and env, tat, rev, nef, vif, vpr, vpu, and tev) encoding 19 proteins. gag & pol contain information needed to make the structural proteins for new virus particles. The rev protein (p19) is involved in shuttling RNAs from the nucleus and the cytoplasm by binding to the RRE RNA element.
Also contained is the human cytomegalovirus (hCMV) which is responsible for the expression of the virus proteins (gag, rev, pol) during translation. The packaging signals and their adjacent signals are removed so the parts responsible for packaging the viral DNA have been separated from the parts that activate them. Thus, the packaging sequences will not be incorporated into the viral genome and the virus will not reproduce after it has infected the host cell.
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Vector Titration
Vector titration typically involves PCR or flow cytometry depending on the presence of a fluorescent protein. For example, mouse NIH 3T3 or human HT1080 cells are infected for 4 hours with limiting dilutions of vector supernate in the presence of polybrene and analyzed for expression of fluorescent protein or detection of vector or packaging sequences by PCR. The titer represents the relative infectivity of the vector as measured on the target cell of choice and is expressed as Infectious Units (IU)/mL. Different target cells, or different infection protocols, may yield different results.
Vector supernate can be concentrated using ultrafiltration. Recoveries (65-90%) vary depending on the envelope used and stability of the viral particle. This technique does not separate empty from functional viral particles. Therefore, at very low titers, concentration may not always yield an improved titer due to the presence of inhibitory empty viral particles.
Packaging cells
Human 293 cells are usually used for packaging, since they can be transfected with efficiencies in the range of 90-100%. If the cells do not transfect well in control experiments using a non-viral GFP vector, it may be worth discarding them and trying a different lot of cells. On occasion, cells can become refractive to transfection. Consequently, use low passage cells for all transfections. Although 293 cells are adherent, they can easily detach from the plate during pipetting, or even by jostling a plate that has reached confluency. For this reason, perform the transfection when the cells are approximately 30% confluent. After 36-48 hours, the cells should reach confluency and be producing the maximum amount of virus. If the cells are fed fresh media, several harvests of virus can be made after the cells reach confluency. One final note: if human cells are used for packaging a RNA hairpin that targets a human sequence, lower viral titers may be experienced if silencing of the targeted gene is detrimental to the cell or packaging process.
Multiplicity of infection (MOI)
Multiplicity of infection (MOI) is the ratio of transfer vector transducing particles to cells. An MOI of 10 indicates that there are ten transducing units for every cell in the well. It is important to note that different cell types may require different MOIs for successful transduction and knockdown of the target gene. For instance, HEK293T cells are highly susceptible to lentiviral transduction (MOI of 5-20) while neuronal cells such as SHSY5Y often require higher MOIs of 10-50.
200 uL containing 10e6 particles = 50,000 particles/uL
- HEK293= 5-10 particles/cell
- SHSY5Y= 10-50 particles/cell
When transducing a lentiviral construct into a cell line for the first time, a range of volume or MOI should be tested.
To calculate:
(Total number of cells per well) x (Desired MOI) = Total transducing units needed (TU)
(Total TU needed) / (TU/ml reported on C of A) = Total ml of lentiviral particles to add to each well
shRNA Transfer Vector Promoters
RNA Polymerase III type 3 promoters (H1, 7SK, U6) are common in vector-based expression because they are permissive expressers, they do not require any additional sequence elements, and have simple termination signals. The most abundant cellular RNAs transcribed from a type III RNA pol III promoter are the U6 small nuclear RNA (which play a crucial role in the processing of premature RNA); the 7SK RNA (a negative regulator of RNA polymerase II elongation factor TEFb); and H1 RNA (a component of RNAse P).
Unlike the majority of RNA pol III promoters, type 3 RNA Pol III promoters do not require any additional sequence elements. In addition, termination of transcription by Pol III occurs at definitive tracts of 4–5 thymidines (T4–5) (Example below), which can be inserted downstream of shRNA coding sequences in order to ensure direct termination. These 2 specific properties enable the U6, 7SK, and H1 promoters to be ideal candidates for in vivo delivery systems of siRNAs where DNA templates can undergo transcription into small RNAs with structural features resembling active siRNA/miRNA.
In summary, Pol III promoters are relatively compact, have high activity, and require a simple poly-U tract for termination.
U6 small nuclear RNA (snRNA) is an essential component of the eukaryotic spliceosomes, is unique in that it is synthesized by RNA polymerase III, while all other U-snRNAs are synthesized by RNA polymerase II. U6 genes are notable for functional upstream regulatory elements which resemble RNA polymerase II regulatory sequence motifs. Tests for both potency and adverse metabolic effects upon primary cells indicate that U6 promoter may exert toxicity relative to H1 or 7SK due in part to higher activity.
U6 snRNA Class III gene initiation
1. SNAPc (SNRNA Activating Protein complex) (also termed PBP and PTF) binds to the PSE (Proximal Sequence Element) centered approximately 55 base pairs upstream of the start site of transcription. This assembly is greatly stimulated by the Pol II transcription factors Oct1 and STAF that bind to an enhancer-like DSE (Distal Sequence Element) at least 200 base pairs upstream of the start site of transcription. These factors and promoter elements are shared between Pol II and Pol III transcription of snRNA genes.
2. SNAPc acts to assemble TFIIIB at a TATA box centered 26 base pairs upstream of the start site of transcription. It is the presence of a TATA box that specifies that the snRNA gene is transcribed by Pol III rather than Pol II.
3. The TFIIIB for U6 snRNA transcription contains a smaller Brf1 paralogue, Brf2. TFIIIB is the transcription factor that assembles Pol III at the start site of transcription. Sequence conservation predicts that TFIIIB containing Brf2 also plays a role in promoter opening.
7SK is an abundant and evolutionarily conserved small nuclear RNA discovered in the 1970s. 7SK is transcribed by RNA polymerase III from one or more genes belonging to a family of interspersed repeats in the mammalian genome (Murphy et al 1984)]. The human 7SK promoter presents a strong permissivity for the nucleotide in the +1 position and recognizes a cluster of 4 or more T residues as a termination signal. The human 7SK promoter is ideal for the production of shRNAs as it can generate high amounts of shRNAs (Czauderna et al 2003, Koper-Emde et al 2004). In a series of experiments aimed to compare the strength of the human 7SK, H1 and U6 promoters, the best silencing efficiencies of various target genes was consistently obtained with a 7SK promoter (unpublished data;Invivogen).
The H1 promoter drives expression of a unique gene encoding H1 RNA, the RNA component of the human RNase P. The H1 RNA gene is transcribed by RNA polymerase III into a small RNA transcript. The human H1 promoter presents the characteristics of being unusually compact. All the essential elements for transcription, that is octamer, Staf, proximal sequence element and TATA motifs, lie within 100 bp of the 5' flanking region. This promoter is highly permissive for the nucleotide at the +1 position which is originally an A. The termination sequence consists of a stretch of 5 thymidines. The cleavage of the transcript after the termination signal occurs after the second uridine generating a 2 nt 3' overhang. The H1 promoter has been successfully used in different plasmids to create siRNAs.
CMV is a RNA Polymerase II promoter. This strong promoter is active in a broad range of cell types and performs better than most pol III promoters under long term selection. However, there are specific reports that CMV driven shRNA do not function properly certain cell types, including CD34+ hematopoietic progentior cells. Ambion offers pSilencer™ 4.1-CMV plasmid vectors designed for gene silencing experiments in a broad range of cell lines (Cat# AM5775).
The CMV promoter is considered to be a stronger promoter than other common RNA pol II promoters used in mammalian expression vectors such as Simian virus-40 (SV40) and Rous sarcoma virus (RSV) (Foecking, 1986). In vivo, RNA pol II is primarily responsible for transcription of mRNA within the cell. The CMV promoter has the advantage of being highly active in a broad range of cell types, and it is suggested to not interfere with other transcription events as may be the case with the RNA pol III U6 and H1 promoters in some situations.
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shRNA Transfer Vector Transfection Reagents
Transient gene silencing using RNAi is critically dependent on highly efficient delivery of the shRNA transfer vector or siRNAs into cells. The two conventional reagent types are Cationic lipid-based and Polymeric formulations. All commercial transfection reagents are proprietary formulations that are competing for market share by claiming certain advantages (ie, broad cell type compatibility, low cytotoxicity, high efficiency).
Cationic lipid
Cationic lipid transfection reagents are suitable for transfecting into a wide variety of dividing cell cultures. Commercial examples include: Lipofectamine / L2000, Dharmafect, iFect, and TransIT TKO. Cationic lipids work by forming lipsomal vesicles that house the siRNA payload and bleb their way through the living cell membrane and into the cytoplasm. The efficiency of this process must be determined in order to have confidence in the knockdown effects. There are numerous commercial sources for transfection reagents for good reason; there are numerous cell types and lipsome structure will influence transfection efficiency in the multitude of experimental cell types that exist.
Polymeric formulations have been developed and optimized for transfection of shRNA plasmid DNA into the nucleus of cultured eukaryotic cells by vendors such as Open Biosystems. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus (Pollard et al 1998). Polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.
shRNA Transfer Vector Transient Transfection Procedure
- In a six well tissue culture plate, grow cells to a 50-70% confluency in antibiotic-free normal growth medium supplemented with FBS.
NOTE: This protocol is recommended for a well from a 6 well tissue culture plate. Adjust cell and reagent amounts proportionately for wells or dishes of different sizes.
NOTE: Healthy and subconfluent cells are required for successful transfection experiments. It is recommended to ensure cell viability one day prior to transfection.
Prepare the following solutions:
NOTE: The optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio should be determined experimentally beginning with 1 μg of shRNA Plasmid DNA and between 1.0 and 6.0 μl of shRNA Plasmid Transfection Reagent as outlined below. Once the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio has been identified for a given cell type, the appropriate amount of shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent complex used per well should be tested to determine which amount provides the highest level of transfection efficiency. For example, if the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio is 1 μg:1 μl, then amounts ranging from 0.5 μg/0.5 μl to 2.0 μg/2.0 μl should be tested.
Solution A: For each transfection, dilute 10 μl of resuspended shRNA Plasmid DNA (i.e. 1 μg shRNA Plasmid DNA) into 90 μl shRNA Plasmid Transfection Medium (serum antibiotic free medium).
Solution B: For each transfection, dilute 1 - 6 μl of shRNA Plasmid Transfection Reagent with enough shRNA Plasmid Transfection Medium to bring final volume to 100 μl.
NOTE: Do not add antibiotics to the shRNA Plasmid Transfection Medium.
NOTE: Optimal results may be achieved by using siliconized microcentrifuge tubes.
NOTE: Although highly efficient in a variety of cell lines, not all shRNA Plasmid Transfection Reagents may be suitable for use with all cell lines.
- Add the shRNA Plasmid DNA solution (Solution A) directly to the dilute shRNA Plasmid Transfection Reagent (Solution B) using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture 15-45 minutes at room temperature.
- Wash the cells twice with 2 ml of shRNA Transfection Medium. Aspirate the medium and proceed immediately to the next step. NOTE: Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency.
NOTE: Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency. For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well.
- For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well.
- Add the 200 μl shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent Complex (Solution A + Solution B) dropwise to well, covering the entire layer.
- Gently mix by swirling the plate to ensure that the entire cell layer is immersed in solution.
- Incubate the cells 5-7 hours at 37° C in a CO2 incubator or under conditions normally used to culture the cells.
NOTE: Longer transfection times may be desirable depending on the cell line.
- Following incubation, add 1 ml of normal growth medium containing 2 times the normal serum and antibiotics concentration (2x normal growth medium).
- Incubate the cells for an additional 18-24 hours under conditions normally used to culture the cells.
Aspirate the medium and replace with fresh 1x normal growth medium.
- Assay the cells using the appropriate protocol 24-72 hours after the addition of fresh medium in the step above.
NOTE: Controls should always be included in shRNA experiments. Control shRNAs are available as 20 μg. Each encode a scrambled shRNA sequence that will not lead to the specific degradation of any known cellular mRNA.
NOTE: For Western blot analysis prepare cell lysate as follows: Wash cells once with PBS. Lyse cells in 300 μl 1x Electrophoresis Sample Buffer (sc-24945) by gently rocking the 6 well plate or by pipetting up and down. Sonicate the lysate on ice if necessary.
NOTE: For RT-PCR analysis isolate RNA using the method described by P. Chomczynski and N. Sacchi (1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159) or a commercially available RNA isolation kit.
Lentivrus Transfection
Prepare mammalian cells growing exponentially and are no more than 70-80% confluent before transduction. Prepare a stock solution of hexadimethrine bromide at 2 mg/ml in water.
Day 1.
- Add 2.0e4 (20,000) cells in fresh medium to the number of wells needed for each construct in a 96-well plate. Duplicate or triplicate wells for each lentiviral construct and control should be used.
- Incubate 18-20 hours at 37°C in a humidified incubator in an atmosphere of 5-7% CO2.
Note: The growth rates of cells vary greatly. Adjust the number of cells plated to accommodate a confluency of 70% upon transduction. Also account for the length of time the cells will be growing before downstream analysis when determining the plating density.
Day 2.
Remove medium from wells. To each well add 110 µl medium and hexadimethrine bromide to a final concentration of 8 µg/ml. Gently swirl the plate to mix.
Note: Hexadimethrine bromide enhances transduction of most cell types. some cells, like primary neurons, are sensitive to hexadimethrine bromide. Do not add hexadimethrine bromide to these types of cells. If working with a cell type for the first time, a hexadimethrine control only well should be used to determine cell sensitivity.
Add lentiviral particles to appropriate wells. Gently swirl the plate to mix. Incubate 18-20 hours at 37°C in a humidified incubator in an atmosphere of 5-7% CO2. Cells may be incubated for as little as 4 hours before changing the medium containing lentiviral particles. Overnight incubation may be avoided when toxicity of the lentiviral particles are a concern.
Note: When transducing a lentiviral construct into a cell line for the first time, a range of volume or MOI should be tested.
Day 3.
Remove the medium containing lentiviral particles from wells. Add fresh medium to a volume of 120 µl to each well. Note: For cell types that do not strongly adhere to the plate, 100 µl of medium may be removed and replaced with 100 µl fresh medium.
Day 4 and forward.
Replace medium every 3-4 days until cells are to be assayed. Cells may be selected and each clone may be expanded to assay for expression of shRNA. A variety of phenotypic, enzymatic, or gene expression assays may be performed. The desired assay should be optimized prior to the high-content screen with both negative and positive controls.
Note: Due to the random integration of the lentivirus into the host genome, varying levels of shRNA expression may be seen with different colonies. Testing a number of colonies will allow the optimal degree of expression to be determined.
shRNA Controls
Negative Controls
- Untreated Cells. Untreated cells will provide a reference point for comparing all other samples.
- Empty construct, containing no shRNA insert; The empty viral particles or DNA are a useful negative control that will not activate the RNAi pathway because it does not contain an shRNA insert. It will allow for observation of cellular effects of the transduction/transfection process. Cells transduced/transfected with the empty control provide a useful reference point for comparing specific knockdown.
- Non-targeting shRNA; This non-targeting shRNA is a useful negative control that will activate RISC and the RNAi pathway, but does not target any human or mouse genes. The short hairpin sequence cotnains 5 base pair mismatches to any known human or mouse gene. This allows for examination of the effects of shRNA transduction/transfection on gene expression. Cells transduced/transfected with the non-target shRNA will also provide useful reference for interpretation of knockdown.
Positive Controls
- Positive reporter vector or lentiviral particles; This is a useful positive control for measuring transduction/transfection efficiency and optimizing shRNA delivery. The GFP Control contains a gene encoding GFP, driven by the CMV promoter. This control provides fast visual confirmation of successful transduction/transfection.
- Positive shRNA knockdown control; This control contains shRNA sequence that targets GFP expression. This shRNA control has been experimentally shown to reduce GFP expression. This control serves to quickly visualize knockdown in cells expressing GFP.
- Positive shRNA knockdown control; This control contains shRNA sequence that targets eGFP expression (GenBank Accession # pEGFP U476561). The shRNA has been experimentally shown to reduce eGFP expression by 90% in C166-GFP mouse fibroblast cells 48 hours post-transduction by mRNA transcript level. This control serves to quickly visualize knockdown in cells expressing eGFP.
Factors Influencing Successful Transfection
Concentration and purity of nucleic acids
Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.
Transfection in serum-free media
The highest transfection efficiencies can be obtained if the cells are exposed to the transfection complexes in serum free conditions followed by the addition of medium containing twice the amount of normal serum to the complex medium 3–5 hrs post transfection (leaving the complexes on the cells). However, the transfection medium can be replaced with normal growth medium if high toxicity is observed.
No antibiotics in transfection medium
The presence of antibiotics can adversely affect the transfection efficiency and lead to increased toxicity levels in some cell types. It is recommended that these additives be initially excluded until optimized conditions are achieved, then these components can be added, and the cells can be monitored for any changes in the transfection results.
High protein expression levels
Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.
Cell history, density, and passage number
It is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.
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text:
Riley nearing Oregon State record
CORVALLIS, Ore. -- Head coach Mike Riley is nearing an Oregon State record as the Beavers face No. 19 UCLA this Saturday.
With a win, Riley would tie Lon Stiner (1933-48) as the school's all-time winningest football coach, with 74 victories.
"The thing I like about it, is that we've been able to be here for a while," says Riley. "We've had a good group of people work at this thing for a long time."
During his 12 years in Corvallis, Riley has taken the Beavers to six bowl games and was named the Pac-10 coach of the year in 2008.
As for this week's matchup, the Beavers defense is in store for a tough test against the second best offense in the country.
UCLA is averaging 622 yards a game under offensive coordinator Noel Mazzone, who joined the Bruins coaching staff this season.
With him he brought his spread offense from his days at Arizona State.
"You know, you got two standard offenses in our league (spread and pro), it's almost divided evenly, says Riley. "It's kind of common for each coaching staff to do what they like to do. We hired, in this conference, a number of guys that are spread coaches."
Riley went on to say that UCLA, Arizona, Washington State and Oregon all primarily run the spread.
For defensive coordinator Mark Banker, it's a good thing.
"To an extent, I really like it because of the fact that you're not just preparing for one team," says Banker. "Now we're preparing for half of our schedule."
To hear more from Riley and Banker, click on the video tabs below.

subset: commoncrawl


text:
Suzanne Hanover / universal studio
We are thrilled that Katherine Heigl is hitting the big screen this summer. Not just because we get to see her at the premieres but because she ditches her boring scrubs for seriously cute and casual on screen clothes. As an entertainment reporter who finds herself Knocked Up Katherine's character Alison stays chic even when switching to maternity wear. The best part is, you can shop while watching the movie. Along with the rest of the cast, all of Katherine's fashion, and looks for less, are available on starstyle.com. Start shopping the look with Katherine's black C&C tank (left) and her costar Leslie Mann's bumble bee necklace and yellow Splendid top. You can see more looks from the movie here!


text:
13 July 2012
The Detroit Tigers made a roster move yesterday that involved assigning reliever Darin Downs to the Toledo Mud Hens. He no longer appears on the team's 40-man roster, so that means he would have had to clear waivers for the team to retain him and send him to the minor leagues.
The move frees up a second spot on the active roster. I had initial thought that Danny Worth would be recalled (and Downs retained after his outstanding performance last week), but that appears to not be the case anymore. Second best odds appeared to be with Worth and relief pitcher Luis Marte returning to the big leagues – and this is probably the most likely scenario still – but it no longer has to be. Downs, a 27 year old who'd been in professional baseball since he was a teenager, was likely out of options* so removing him from the 40-man roster after placing him on waivers would have been the only way to keep him in the system.
*Option year info isn't published publicly anywhere that I know of, so it can be difficult to determine on the fly.
My first question about this is why? Why do you call up an emergency pitcher for six games that isn't already on the 40-man roster if you know you're going to immediately burn him? I guess that's why he's in the system, but the team probably could have called up Luke Putkonen, Thad Weber, or even Adam Wilk (guys already on the 40-man with option years remaining) if they didn't want to risk losing a player. It all worked out for the best though. Downs was fantastic in his 4.1 innings of work (seven strikeouts, one hit, and one walk), and the club still gets to keep him in the minor leagues.It would seem strange to automatically oust Downs, who's been fantastic all year in AAA and so good in his Tigers debut, for Marte who, besides having a pretty good ERA, hasn't really set the world on fire with his peripheral numbers in Detroit. As I've said this could really be the case, but the extra spot on the 40-man roster means the organization isn't forced to go this route.
Here are three additional non-40-man possibilities for the immediate to not-so-distant future:
Ben Guez, OF: Guez is a guy that's not often considered along with the prospects in the system but, like Andy Dirks, he could turn into an adequate third-fourth-fifth outfielder in the big leagues. His minor league numbers show good on-base skill (.348 minor league career OBP), and some decent extra base power. He likely wouldn't bring a ton of pop to a big league lineup.
Nick Castellanos, 3B/OF: It freaks me out a lot that I'm even typing this, but it's hard for me to explain why the organization would have him play in the outfield if they're not contemplating calling him up sooner rather than later. I would rather them wait at least until next spring to give him his first shot (I don't like his K/BB ratio in AA so far), but he's definitely the "next big thing" as far as Tigers hitters go. I just hope they don't abandon the third base thing right now because he brings a lot more value to 2015 and beyond at the hot corner than he would in the outfield.
Bruce Rondon, RP: This is another very unlikely scenario. Rondon is probably the top relief/future closer prospect in the system, and he's been lights out this year. Like Castellanos, he was only recently promoted to Erie (from Lakeland). He's pitched well at both levels, but I have a hard time seeing him make the big jump to the bigs right now. He still needs to work on his control, and the Tigers bullpen has been quite good for the better part of two months; it doesn't seem like they would need to rush a prospect there.
Matt Snyder is the editor of The Tigers Den. He can be reached on Twitter @snyder_matthew.


text:
I hope he has sufficient evidence, cause word of mouth will not justify a $50 claim let alone a $600M claim.
nothing to do with jayz, wtf
Well…if there is a patent on the idea….there's no getting around it. I don't think even if they do settle he will get anything near $600M, but if he did register the trademark he will get a good amount. Lawyers can protect you, but you or someone you enlist for the job need to keep on top of them to keep all your ducks in a row.
He wants that jay n bey type money.
"…but his estimated net worth is a tad bit shy of Dr. Cassagnol's asking price by $100M." Where do these writers get their information? Jays networth is over 400m.


text:
kirtu wrote:narraboth wrote:The best way for some people probably is to drag out lama's corp out and put on an iron bed, and measure it every hour.
This is what modern people want and it goes back to what Pema Ridgzin mentioned.
But it is a Nyingma forum, so I guess some people would be benefit. At least I hope.
I have never wanted to use this photo to convince some christians.


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Well when I take my son to the game I'm very aware of the people around me.
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Well when I take my son to the game I'm very aware of the people around me.
There are two types of quarterbacks in the league: Those whom over time, the league figures out ... and those who figure out the league.
OK so if you should eb able to figure it out don't you take her inability to figure it out then as a sign the guy was not drunk?
Maybe the guy was drunk I don't know but the writer admits she didn't know, and she admits she wasn't interested in an apology. I understand her being upset, I don't understand the desire to write an article about it.Really, we had no good answer. Everyone who has heard the story immediately says, the guy was drunk. To be honest, checking the guys inebriation status was not top of our mind.
Anywho, the kid had some vague recollection of them wrestling. I have no idea how the parents have no idea.
Also let's talk about this quote
After saying this is not the first time this season an innocent fan has been wounded...she goes from talking about the Pacers to....the Colts? Do they share the same season now? That is some wiley writing I tell you what.The most frustrating thing, as my son quickly deduced, was that this all could have been avoided, had the guy and his friend used some commonsense. Nor is it the first time this season that an innocent fan has been wounded by another ticket holder.
Heck I could say I am the freakin Pope and I would baptize him right then and there in a beer vat but I would be lying to you.
Last edited by Gamble1; 01-16-2014 at 03:32 PM.
I guess I can share another reason I'm a bit on the guy's side. The game against the Nets I got tickets for our whole family to go to the game. It was pretty much sold out by the time we knew they'd all be here, so I had to get some of the tickets about halfway up section 223. I got a custom jersey for my grandson, and it was delivered to me about the middle of the second quarter. I headed up to give it to him.
They were sitting in the middle seats of the row, and I had to sidle across a bunch of people to get to them during a timeout. The people in the intervening seats tried to move but there was still very little room for my feet to move past. As I got to my family, I tripped over one guys leg and came within an inch of pitching down into the seats below me. One guy had to grab me to keep me from falling. If I'd had a drink in my hand instead of a jersey, it would have been all over the place.
I'd had a beer at that point. I don't think I was drunk, I was certainly able to maneuver every other place I'd had to go that night, but those seats can be downright dangerous getting into and out of.
A bird in the hand is worth two in the bush.
Or throw in a first-round pick and flip it for a max-level point guard...
...still making me chuckle...
I'm not proud of that, I'm just sayin'...
And she should be biased, I just think it was really amateur and unprofessional by both her and the star to write a whole article about the event. I honestly don't have any problem with how she reacted in the heat of the moment, but I feel like she should have taken a step back before writing the article.
I've had a drunk slam into me while running down the sidewalk. I don't blame the city of Indianapolis for that. I'm not avoiding all sidewalks because of it either. Things happen. That's life. This woman's child is going to get hurt. He is going to get hurt because of somebody else. That doesn't mean you go out and write an article blaming people for it. Accidents happen.
I've probably almost fell at least once at every game I have ever gone to. Granted I have a bum knee. But what if that were the case with this guy? Would all this vitreous hate turn to sympathy?
It might be time to apply for those special seats if you have fallen nearly every time you have gone. I know if I had a choice to watch a game I would much rather be in the handicap row than up in the nose bleeds.
Accidents and drunkenness both happen all the times at Pacers games.
We had beer spilled all over the back of us on Tuesday night in the Club Level by the obnoxiously drunk guys in the row behind us. Had my niece or nephew been with me, I probably would have alerted the usher. Their language was pretty offensive and I am not at all easily offended. The staff/ushers do a great job of dealing with things like this, but they can't see everything.
I have been to hundreds of games. I have accidentally fallen twice. It's pretty easy to do. Accidents happen.
Saying all of that, I think it's pretty ridiculous that this had to be published by the Star. It seems more like a Facebook/Tumblr rant than anything.
Passion. Pride. Patience. Pacers
I think it was a serious helpful suggestion. The handicapped seats aren't just for wheelchairs, and if you have a bad knee that makes it even harder for you to get around in the narrow seats you should take advantage of it.
A bird in the hand is worth two in the bush.
Or throw in a first-round pick and flip it for a max-level point guard...
They don't ask many questions. As long as the seat is available, you can request it.
A bird in the hand is worth two in the bush.
Or throw in a first-round pick and flip it for a max-level point guard...
Because she just happens to work for The Star, there was an opening and she knew this would spark interest.
Why? I see someone fall down the stairs at Bankers life pretty much every game. I've seen numerous people fall down onto people below. It has nothing to do with being drunk, and everything to do with the incredibly steep stairs. Mostly I've seen sober women fall (wearing heels) but I've seen just about every demographic lose it on those stairs. Hell, I have a friend who won't even sit in the upper deck because it's to steep and gives him vertigo.
Also, the lady who wrote this story is rude and obnoxious. She immediately judges the people involved, ignores their apologies, and blames everyone without taking into account any circumstances. She is essentially making the world a worse place for everyone involved. She needs to grow up and realize that accidents happen. If she can't do that, then she should just stay at home. Imagine what she would write about the person who spins out on ice and crashes into her car with her son in the back??
Danger Zone
Poor kid is now getting punished because of an accident that he didn't even cause. She is like the Pacer Game Nazi. "No Pacers for you!"
Helicopter Mom
A bird in the hand is worth two in the bush.
Or throw in a first-round pick and flip it for a max-level point guard...
The reason for the article and the style of the writing makes perfect sense now.
I find it strange that she is thinking about why did it happen. As if it was on purpose or some plot against her and her family. Seems like a strange question. Either it was an accident, or the guy was being a jerk. Forget it and move on. And how can you be sure it won't happen again. Well chances are it won't. but that is life


text:
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More than 39 years of innovations, visions and creations - paired with emotions.
Challenges are our incentive.
Visionary mobility concepts, innovative drives, sustainability, comprehensive network and stated competences in world wide communications are part of our core business.
"You may say I'm a dreamer, but I'm not the only one, I hope some day you'll join us, and the world will live as one"
John Lennon / Beatles / "Imagine"
"If you can dream it, you can do it"
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"First they ignore you, then the laugh at you, then they fight you and then you win"
Mahatma Gandhi
"Always aim for the moon, even if you miss, you'll land among the stars"
Frank M. Rinderknecht
Founder and CEO of Rinspeed Inc.


text:
MIAMI - As the police union continues to negotiate with Miami-Dade County over contracts, a pricey issue raised in a Local 10 investigation is on the police director's radar.
Each year, $1.4 million are being spent on gas and maintenance to allow Miami-Dade police officers to drive their patrol cars to their homes outside of Miami-Dade County at a time when the budget needs to be cut.
In some cases, officers drive across the state to Naples or up two counties to Palm Beach.
"We are certainly, I'm telling you, reviewing and refining the process right now," said Miami Dade Police Director James Loftus.
As Local 10 reported, three Miami-Dade police officers live in and drive to Collier County, which is 121 miles one way and 242 miles round-trip, resulting in 1,210 miles per week. That is $10,000 per year for gas for one officer in one vehicle to get to and from work.
Ten officers drive through Broward to Palm Beach County.
"All of these things are being revisited. When I talked about revisiting the policy, that's what I'm doing. It's within my purview," Loftus said.
The police budget needs to be drastically cut as the police union and the county continue to negotiate.
"Believe me. I'm telling you we are banging away on $20,000, $10,000, $5,000 --whatever it takes to narrow this gap," Loftus said.
Loftus originally proposed a $25 per week, across-the-board fuel surcharge for officers who have take-home vehicles, but many, including the mayor, said that those who live outside Miami-Dade County should pay more.
"I'm a guy who would be paying 25, 50 or 75 bucks a pay period, and quite frankly, I'm fine with that. I balance that with (what) would it cost me to buy a car and drive to work and go to all the places I go on a daily basis, and I sa, 'Hey, that's easy to swallow,'" Loftus said.
The Police Benevolent Association and Miami-Dade County continue contract negotiations Nov. 1.
Copyright 2011 by Post-Newsweek Stations. All rights reserved. This material may not be published, broadcast, rewritten or redistributed


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text:
Evaluation of Patient Reported Outcome Instruments in Celiac Disease Patients
This study has been completed.
Alvine Pharmaceuticals Inc.
Information provided by (Responsible Party):
Alvine Pharmaceuticals Inc.
ClinicalTrials.gov Identifier:
First received: March 20, 2012
Last updated: October 9, 2013
Last verified: October 2013
- Full Text View
- Tabular View
- No Study Results Posted
- Disclaimer
- How to Read a Study Record
This is an evaluation of celiac-specific patient reported outcome instruments in celiac disease patients.
|Celiac Disease
|Study Type:
|Study Design:
|Time Perspective: Prospective
|Official Title:
|Clinical Evaluation of Three Celiac Disease-Specific Patient Reported Outcome Instruments in Established and Newly Diagnosed Celiac Disease Patients
Resource links provided by NLM:
Genetics Home Reference related topics: celiac disease
MedlinePlus related topics: Celiac DiseaseU.S. FDA Resources
Further study details as provided by Alvine Pharmaceuticals Inc.:
Primary Outcome Measures:
- Change in symptoms using patient reported outcome instruments [ Time Frame: 8 and 12 weeks ]Sensitivity of the patient reported outcome instruments to detect change over time in celiac disease symptoms with and without a gluten challenge
Secondary Outcome Measures:
- Safety - incidence of adverse events [ Time Frame: 8 and 12 weeks ]Safety will be evaluated by incidents of adverse events including clinical significant laboratory evaluations and serious adverse events
|Study Start Date:
|March 2012
|Study Completion Date:
|September 2013
|Primary Completion Date:
|September 2013 (Final data collection date for primary outcome measure)
Gluten challenge
Gluten containing or gluten-free study food in established celiac disease patients
Observation in newly diagnosed celiac disease patients
|Ages Eligible for Study:
|18 Years to 70 Years (Adult, Senior)
|Sexes Eligible for Study:
|Accepts Healthy Volunteers:
|Sampling Method:
|Non-Probability Sample
Study Population
Biopsy proven celiac disease patients
Inclusion Criteria:
- History of biopsy proven celiac disease
- Adherence to a gluten-free diet (established patients only)
- TG2 negative (established patients only)
- TG2 positive (newly diagnosed patients only)
- Signed informed consent
Exclusion Criteria:
- History of IgE-mediated reactions to gluten
- Significant laboratory abnormalities
- History of untreated or GI disease
- Positive pregnancy test
- Any medical condition which could adversely affect study participation
Contacts and Locations
Choosing to participate in a study is an important personal decision. Talk with your doctor and family members or friends about deciding to join a study. To learn more about this study, you or your doctor may contact the study research staff using the Contacts provided below. For general information, see Learn About Clinical Studies.
Please refer to this study by its ClinicalTrials.gov identifier: NCT01560169
Please refer to this study by its ClinicalTrials.gov identifier: NCT01560169
|United States, California
|University of California, San Diego
|La Jolla, California, United States, 92037
|Kaiser Permanente Southern California, Allergy Department
|San Diego, California, United States, 92111
|United States, Colorado
|University of Colorado, Denver
|Aurora, Colorado, United States, 80045
|United States, Illinois
|University of Chicago Celiac Disease Center
|Chicago, Illinois, United States, 60637
|United States, Massachusetts
|Beth Israel Deaconess Medical Center
|Boston, Massachusetts, United States, 02215
|United States, Minnesota
|Mayo Clinic
|Rochester, Minnesota, United States, 55905
|United States, New York
|Columbia University
|New York City, New York, United States, 10032
|United States, Ohio
|Cleveland Clinic
|Cleveland, Ohio, United States, 44195
|United States, Pennsylvania
|Thomas Jefferson University
|Philadelphia, Pennsylvania, United States, 19107
|Canada, Alberta
|University of Alberta
|Edmonton, Alberta, Canada, T6G 2X8
Sponsors and Collaborators
Alvine Pharmaceuticals Inc.
|Study Chair:
|Daniel Adelman, MD
|Alvine Pharmaceuticals Inc.
More Information
Keywords provided by Alvine Pharmaceuticals Inc.:
Additional relevant MeSH terms:
ClinicalTrials.gov processed this record on September 21, 2017
|Responsible Party:
|Alvine Pharmaceuticals Inc.
|ClinicalTrials.gov Identifier:
|NCT01560169 History of Changes
|Other Study ID Numbers:
|Study First Received:
|March 20, 2012
|Last Updated:
|October 9, 2013
Keywords provided by Alvine Pharmaceuticals Inc.:
Celiac Disease
Coeliac Disease
Celiac Sprue
Keilakia Disease
Additional relevant MeSH terms:
Celiac Disease
Malabsorption Syndromes
Intestinal Diseases
Gastrointestinal Diseases
Digestive System Diseases
Metabolic Diseases
ClinicalTrials.gov processed this record on September 21, 2017


text: Our Fab Gift Guides are well underway. Next up on our list is the GQ dad — or the dad you want to make more slick! For him, we have picked an array of items to make him the most dapper dad on the block. Take this snowflake sweater by Ralph Lauren; it's perfect for his next ski adventure. He can pack it in Kenneth Cole's wool duffel, along with these hot Tom Ford aviators. Enter for seven more chic gift ideas for dad.


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Sign into our member site to view your information online.Forget your username or password? Not registered? Register here
aldesleukin (generic name)
Proleukin (brand name)
It is usually used for advanced renal cell cancer or for advanced melanoma
(al des LOO kin)
Table of Contents
Top Learning Centers(Recursos en Español)
What is this medicine?ALDESLEUKIN, IL-2 (al des LOO kin) is a chemotherapy drug. It is usually used for advanced renal cell cancer or for advanced melanoma. It is sometimes used for other cancers.
What should I tell my health care provider before I take this medicine?They need to know if you have any of these conditions:
How should I use this medicine?This medicine is for injection into a vein but can also be injected under the skin. It is given by a health care professional in a hospital or clinic setting.
Talk to your pediatrician regarding the use of this medicine in children. Special care may be needed.
What if I miss a dose?This does not apply.
What may interact with this medicine?
What should I watch for while using this medicine?Your condition will be monitored carefully while you are receiving this medicine.
Visit your doctor or health care professional for regular checks on your progress. You will need frequent blood checks.
Try to avoid people who are sick. If you get a cold or other infection while receiving this medicine, call your doctor or health care professional. Do not treat yourself. The medicine may decrease your body's ability to fight infections.
The medicine may make you feel generally unwell. This is not uncommon. Tell your doctor or health care professional about any side effects or problems you develop.
What side effects may I notice from receiving this medicine?Side effects that you should report to your doctor or health care professional as soon as possible:
Side effects that usually do not require medical attention (report to your doctor or health care professional if they continue or are bothersome):
Where should I keep my medicine?This drug is given in a hospital or clinic and will not be stored at home.
Remember, keep this and all other medicines out of the reach of children, never share your medicines with others, and use this medication only for the indication prescribed.


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Encouraging and supporting citizen journalism in Nepal
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Birendranagar, surkhet ., Nepal
chandigarh, India
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text:
I finally got around to trying Sketchup. I'm a Blender user so I didn't know if it was worth looking at or not. I'm very impressed. It's the most intuitive modeler I've ever used. They have about 4 twenty minute videos to bring you up to speed. It's a cad program, actually, so I can use it for some building projects I do once in a while, plus, I'm going to see what the export is like because I'll probably use it for architecture instead of Blender. They've actually changed the export to collada so there shouldn't be much fuss. It used to be a zipped collada that had to be renamed.
You should use 3 or 4 programs. Scetchup for too low poly or for levels. Blender/3D max/Maya for characters, vehicles etc., Zbrush for High-poly.
Yes, I agree. I haven't tried Zbrush, yet, though. I've already run against a problem with Sketchup, though. It doesn't do a very good dae export. It crashes Blender a lot. Don't know if it's just my computer or what. I've been using a plugin script and doing dxf exports with it right now. I have to texture it in Blender that way. The models are enormous in Blender too. They have to be scaled way down.
IMO there's no such thing as a good Collada exporter/importer. The format almost floats on dreams of being a universal standard. Almost no app properly implements it, or greatly abuses it by using extensions instead of the standard elements. Blender in particular does not fully implement the standard, so it's not unusual to cause problems when you import from another app. I wouldn't be surprised if SketchUp implemented their own version of Collada either. I remember reading a blog post from a Google employee about using Collada for web presentations and seeing his very narrow view of the Collada format. It was because of that I ended up writing my own importer instead of using Google's.
I haven't used ZBrush, but the sculpting features available in Blender work fairly well from what I have done with it. Personally I'd rather work with nurbs, but it totally depends on what you're modelling. I don't even pretend to be able to model characters, so playing with squishy polygons doesn't cut it for me.
IMO there's no such thing as a good Collada exporter/importer.
Yeah. I've found there's one standard worth using and it's fbx. All the others are deficient in one way or another. They don't include camera, they allow too much variation, they don't have bone animation, etc, etc. It might not be open source, but it's open enough and it works. Blender is horrible about keeping up their exporters. They don't seem to care if they work or not. The md2 has been broken since version 2.30. I know a lot of people that would still like to use it. It comes in handy. But I'm pretty sure this is Google because I tried a free convertor from whoever makes fbx, can't think of their name, but it just showed an empty model when I imported it converted to obj. It was just a simple model, too. Some of them work, some don't. The dxf seems pretty consistent.
I've seen the FBX format once in an XNA skinning demo for WP7. I found it a bit "texty" for my liking. Surprisingly the file loaded quite fast on the phone, but I figure if you had to load several dozen files in that format you would be sitting around for quite some time. One format that is renouned for excellent support, albeit basic in features, is the OBJ file. If you just need geometry storage, that's a solid format to work with. And if portability is not an issue, you might as well role your own format. The blender plugin I posted in code forum here is the format of choice I use today. It works very nicely and I don't regret putting the effort into it. I can actually work with keyframe animations instead of that horrid BVH format.
You can save fbx in either text or binary. The binary is pretty fast. Yeah, obj works good for simple static models. In Unity, I think it's saving the models in it's own format once the game is written unless they are specifically saved. I could be wrong about that, though. The trouble with roll your own is it takes time away from game writing and it's one more individual format that works for one specific purpose and doesn't share well. For a while I learned OpenGL, and even wrote a simple exporter for it from Blender, but I found it was too much trouble keeping up with everything. With an engine, that falls on someone else's shoulders and I can just write games which is what I'm interested in. All the little incompatibilities that crop up, that turns into a full time job. I want to just write a game and move on. Learning is fun, but it's enough for me to learn modeling and engine functions.


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LIVERMORE — From the outside, it resembles any other suite in a nondescript industrial park.
But inside, its shelves stocked with barrels of liquid gold bearing names like AWOL Pale Ale, Boot IPA, and Bazooka Brown, Uncle Sam's Misguided Brewery is clearly a twist on an age-old craft.
"We don't have loads of money,"said co-owner Josh Laine, a Marine sergeant who was "shot, stabbed and blown up" during two tours in Iraq. "It's such a small scale that we can be hands-on and give our beers that quality that people are looking for."
After years creating home-brewed concoctions, Laine and some military veteran beer-making buddies sold kegs at holiday parties to launch the USMB Tap House in March, the latest in a string of craft breweries and taprooms to open in Livermore.
An offshoot of the award-winning Valor Winery in Livermore — which Laine started in a converted school bus with a fellow Marine in 2007 — the veteran-owned and operated microbrewery is not only looking to hire veterans, but also donates a portion of profits to non-traditional rehabilitation programs for returned soldiers, like wilderness retreats, yoga, sailing, and equine therapy.
The main beneficiary is the Vets and Vines Foundation, a nonprofit Laine founded to teach viticulture and farming in the vineyards. Its cornerstone project is Valor Ranch, a 290-acre Manteca farm with grapevines, gardens, stables, housing and a wellness center to train veterans in sustainable living techniques. The project is in the first phase of planting.
According to co-owner Fara Barnes — a former Defense Department contractor and veteran of the Air Force and Air National Guard — in a "saturated" wine industry, Valor needed a new platform to create jobs and educate civilians on the stigma facing returning soldiers.
"We have faced prejudice being a veteran," Barnes said. "We have both gone through rehabilitation ourselves and we're both very passionate about helping our brothers and sisters. When you are that type of a person, it becomes very important to bring people on board and lift people up as you're lifting yourselves up."
The winery and brewery combined have hired nearly 60 veterans in full or part-time roles, Laine said. About 200 more have been assisted through vocational training or other resources. Uncle Sam's has a staff of 34 paid employees (full-time and independent contractors), 16 veteran and 10 civilian volunteers, and five intern brewers looking to be hired on when production increases.
Army veteran Gordon Yardy was in dire financial straits and struggling to find a home when he started working in the winery's warehouse, cleaning machinery and prepping bottles for filling.
"They helped me with getting my benefits," Yardy said. "Through my work with Valor I was able to get back on my feet."
Now in the construction business, Yardy continues to help out at the brewery when needed, appreciative of bonds he forged laboring alongside his brothers-in-arms.
"Its helped me tremendously," he said. "I made some good friends and met some good people."
According to VA Palo Alto Health Care System spokesman Michael Hill-Jackson, veteran-owned businesses like Valor and Uncle Sam's are vitally important to veterans attempting to readjust to society with "invisible discrimination" from employers.
"The hardest thing is to get (veterans) into jobs where that professional atmosphere doesn't suit them and that stigma is present," Hill-Jackson said. "It's good to be around people who have had similar experiences."
To further encourage a spirit of brotherhood, profits from the brewery and winery — half of online wine sales go to the purchaser's program of choice — support seven nonprofit and cause partners like Wounded Wear and Huts for Vets, a wilderness rehabilitation program based in the Colorado Rockies. This fall, Valor and Uncle Sam's will expand to offer rehabilitation kits and a hunting and fishing series on ranches in Northern California and Texas, the latter working with Wayne Kyle, whose son Chris, a former Navy SEAL and author of the bestseller "American Sniper," was shot and killed at a Texas gun range in 2013. An Iraq War veteran diagnosed with post-traumatic stress disorder, Eddie Ray Routh, has been charged with Kyle's murder.
Wayne said he is "100 percent" behind the efforts of Laine and Barnes, in hopes they will create public awareness of veterans' issues, and educate wounded warriors in what resources are available.
"These programs like (Barnes) has are extremely important," Kyle said. "Our men and women voluntarily put themselves in harm's way. They sacrifice a lot, more than most people will even realize … There are those who are suffer greatly internally where you can't see."
Over Memorial Day weekend, Uncle Sam's brewed a Valor Double IPA in Kyle's honor, with half the profits going to the Chris Kyle Memorial Roping and American Valor Foundation, organizations founded by the Kyle family.
Currently able to produce up to 288 barrels per month, Uncle Sam's is among the finalists for appearing on the ABC reality TV show "Shark Tank," and is on the hunt for investors to increase production, hire full-time employees and expand their beers into bars, retailers and restaurants.
"Every time people share our story and our mission, they truly do decrease the amount of time it takes for wounded warriors and veterans to rehabilitate," Barnes said.
Contact Jeremy Thomas at 925-847-2184. Follow him at Twitter.com/jet_bang.
For more on Valor Winery and Uncle Sam's Misguided Brewery, visit www.valorwinery.com, www.usmbtaphouse.com, or visit their pages on Facebook. The USMB Tap House is at 2111 Research Drive Suite #4 in Livermore.


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The .75/1 Clorox coupon is gone too 🙁
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Call it the return of the default deniers. To these conservatives, hitting the debt ceiling is no Armageddon, and the idea of "default" is just a scare tactic – and they claim not to be frightened.
"No treasury secretary or president would actually default on the debt, even if they couldn't borrow more money," Toomey (R-Pa.) told POLITICO.
Toomey calls President Barack Obama's warnings of imminent default an excuse to "keep spending and never address the underlying problem."
Scalise, the new chairman of the Republican Study Committee, a huge bloc of House conservatives, has been making the same case. "President Obama is the only person in town right now saying the federal government will not meet its obligations; that's just not the case," he told POLITICO.
Reprising a role they played in the 2011 debt ceiling standoff, the lawmakers are arguing that U.S. creditors will continue to be paid if the government crashes into the debt ceiling — and that what's really at stake is a government shutdown, not a debt default…
"We collect money every day; it just depends on how you spend it," rising Republican star Jason Chaffetz of Utah, a prioritization supporter, said in the House basement Monday. "We're just talking about slowing down government spending."


text:
Marketing Information
The ADATA microSDHC Memory Card Class 10 brings you unrivaled data transfer performance, providing a total upgrade solution that is ideal for the latest generation of mobile phones. At only a quarter the size of a standard SD card, the ADATA microSDHC Class 10 boosts storage capacity and enhances transfer speeds to a minimum of 10 MB/s. This revolutionary little card allows you to take high-resolution photos or record full HD video with your mobile phone-anytime, anywhere.
General Information
|Product Type
|MicroSD High Capacity (microSDHC)
|Manufacturer Part Number
|Manufacturer Website Address
|A-DATA Technology Co., Ltd
|Product Model
|Product Name
|32GB microSD High Capacity (microSDHC) Card
|Package Type
|Brand Name
Technical Information
|Storage Capacity
|Number of Memory Cards
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|Class 10
Physical Characteristics
|Weight (Approximate)
|0.01 oz
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|MicroSD High Capacity (microSDHC)
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This product is shipped only within Canada and Warranty valid for Canada only.
Computer Valley makes every effort to provide accurate specifications, descriptions, images and price on all products that are found on www.computervalley.ca
However for complete and updated descriptions, compatibilities, specifications and actual photo we recommend our clients to consult with manufacturer's websites or simply call us for proper clarifications. Computer Valley is not responsible and will not accept returns for items purchased that are not compatible with your system. Please ensure that you have verified compatibility and actual photo with your system by visiting the manufacturer's web site prior to purchase or give us a call to make sure what you are purchasing.
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Computer Valley wants to provide you with the best and fastest possible service. We try to ship in stock orders placed before 2:00pm EST the same day. For in stock orders placed after 2:00pm EST they are usually shipped the next business day. However there are always exceptions. Please contact us prior to placing your order if you need your purchase by a certain date.
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Make your installation as clean and professional as possible with these extension cords featuring a flat 360° rotating plug.
by Featherstone
on 3/21/2015
I've used similar types of short adapter plugs, but this flat rotating plug head is very cleaver and works extremely well in tight places.
by mspost
on 1/5/2015
This is a simple product for those hard to reach outlets. I used mine to plug in my washer and still be able to use the other outlet. This cord is well made and the rotating head is perfect.
by fredstrade
on 3/5/2013
This product works well when we try to get desks and furniture as close to the wall as possible
by carhard2
on 9/17/2012
You plug it in the wall and plug your device into it. If you find this complicated, then return to your cave and keep saying "OOOOHHHHHMMMM" while pondering whatever Pinkie is pondering.
It works
by barbacci
I use these exclusively in tight places (book cases, furniture etc.) Never once has this product failed me.
Highly recommended.
by Utopiaprime
on 4/14/2012
Well made cables, right color that I needed and good length. No more bulky power adapters' bulging out and pushing against the furniture. Power cables attached to the rotating plug extension cables are running where I want them to go.
by dministries
on 3/29/2012
I haven't actually used these yet (I bought several), but they seem well made, and they are rated for a full 15 amps, in spite of the rotating plug, which apparently uses a sliding connection rather than stranded wiring. I hope they stand up under extended use.
by ramsay
on 2/28/2012
These are handy little cords. They are useful where a desk comes right against a wall outlet, of when you have to plug a power brick into a crowded strip.
by jstephens203
on 1/26/2012
This product is designed well and works great for my situation...
by mgdion
on 8/31/2011
If you're looking for a rotating plug that lies flat against the wall and extends your electric needs around a foot or so, you've found it. Not sure what else one could expect from this device, but for the special price MCM had them for a few weeks ago, it was a no-brain purchase.
I've ordered several times from MCM and have always been very satisfied with their products, service and shipping.


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- Fact Sheet
New Americans in Maine
The Political and Economic Power of Immigrants, Latinos, and Asians in the Pine Tree State
Immigrants, Latinos, and Asians account for significant and growing shares of the economy and population in Maine. Immigrants (the foreign-born) make up 3.4% of the state's population, and more than half of them are naturalized U.S. citizens who are eligible to vote. Moreover, Latinos and Asians (both foreign-born and native-born) wield $928 million in consumer purchasing power, and the businesses they own had sales and receipts of $442.5 million and employed more than 3,000 people at last count. As the economy continues to grow, Maine can ill-afford to alienate an important component of its labor force, tax base, and business community—especially since the state's population is aging rapidly and a growing number of retirees are depending on a declining number of workers.
Immigrants and their children are significant shares of Maine's population and electorate.
- The foreign-born share of Maine's population was 3.4% in 2013, according to the U.S. Census Bureau. Maine was home to 44,687 immigrants in 2013.
- 56.7% of immigrants (or 25,351 people) in Maine were naturalized U.S. citizens in 2013—meaning that they are eligible to vote.
- Unauthorized immigrants comprised less than 0.2% of the state's population (or fewer than 5,000 people) in 2012, according to a report by the Pew Hispanic Center.
- 3.1% (or 24,030) of registered voters in Maine were "New Americans"—naturalized citizens or the U.S.-born children of immigrants who were raised during the current era of immigration from Latin America and Asia which began in 1965—according to an analysis of 2012 Census Bureau data by the American Immigration Council.
Maine is home to an appreciable number of Latinos and Asians.
- The Latino share of Maine's population grew from 0.7% in 2000 to 1.4% (or 18,432 people) in 2013. The Asian share of the population grew from 0.7% in 2000 to 1.1% (or 14,059 people) in 2013, according to the U.S. Census Bureau.
- In Maine, 79.5% of children with immigrant parents were U.S. citizens in 2009, according to data from the Urban Institute.
- In 2009, 97.2% of children in Latino families in Maine were U.S. citizens.
Immigrant, Latino, and Asian entrepreneurs and consumers add hundreds of millions of dollars and hundreds of jobs to Maine's economy.
- The 2014 purchasing power of Maine's Latinos totaled $424 million—an increase of 650% since 1990. Asian buying power totaled $504 million—an increase of 522% since 1990, according to the Selig Center for Economic Growth at the University of Georgia.
- Maine's 1,107 Asian-owned businesses had sales and receipts of $279.3 million and employed 2,550 people in 2007, the last year for which data is available. The state's 979 Latino-owned businesses had sales and receipts of $163.2 million in 2007, according to the U.S. Census Bureau's Survey of Business Owners.
- From 2006 to 2010, there were 2,711 new immigrant business owners in Maine. These businesses had total net business income of $120 million (3.3% of all net business income in the state), according to Robert Fairlie of the University of California, Santa Cruz.
- In 2010, 3.2% of all business owners in Maine were foreign-born, according to the Fiscal Policy Institute.
Immigrants are important to Maine's economy as workers and taxpayers.
- Immigrants comprised 3.6% of the state's workforce in 2013 (or 25,333 workers), according to the U.S. Census Bureau.
- Unauthorized immigrants comprised less than 1% of the state's workforce (or fewer than 5,000 workers) in 2012, according to a report by the Pew Hispanic Center.
- Latinos in Maine paid $41.9 million in federal taxes and $22.8 million in state/local taxes in 2013, according to the Partnership for a New American Economy. In particular, foreign-born Latinos paid more than $19 million in federal taxes and $7.9 million in state/local taxes in 2013.If all unauthorized immigrants were removed from Maine, the state would lose $137 million in economic activity, $60.9 million in gross state product, and approximately 1,080 jobs, even accounting for adequate market adjustment time, according to a report by the Perryman Group.
- The federal tax contribution of Maine's Latino population included $31.8 million to Social Security and $7.4 million to Medicare in 2013. In particular, foreign-born Latinos contributed $11.7 million to Social Security and $2.7 million to Medicare in 2013.
- The importance of immigrant workers is growing as Maine's population becomes older. Over the next two decades, the ratio of seniors (age 65 and older) to prime-working-age adults (age 25 to 64) in Maine will increase by 93%, according to a study by the University of Southern California.
Unauthorized immigrants pay taxes.
- Unauthorized immigrants in Maine paid $3.7 million in state and local taxes in 2010, including $2.8 million in sales taxes, $464,000 in state income taxes, and $432 thousand in property taxes, according to data from the Institute for Taxation and Economic Policy.
- Were unauthorized immigrants in Maine to have legal status, they would pay $4.5 million in state and local taxes, including $2.9 million in sales taxes, $1.1 million in state income taxes, and $451,000 in property taxes.
Immigrants contribute to Maine's economy as students.
- Maine's 1,198 foreign students contributed $44.1 million to the state's economy in tuition, fees, and living expenses for the 2013-2014 academic year, according to NAFSA: Association of International Educators.
- Foreign students also contribute to innovation in Maine. In 2009, "non-resident aliens" comprised 20.8% of master's degrees and 41.7% of doctorate degrees in science, technology, engineering, and mathematics (STEM) fields, according to the Partnership for a New American Economy.
Immigrants have helped revitalize Lewiston.
- Roughly 3,500 Somali migrants came to Lewiston between 2001 and 2007, and now comprise 10% of the town's population—the highest concentration of Somalis in America, according to a report by the United Nations Development Program.
- Although enrollment at the University of Maine has declined statewide since 2002, the student population at its Lewiston campus increased 16% between 2002 and 2007.
Immigrants excel educationally.
- The number of immigrants in Maine with a college degree increased by 53.7% between 2000 and 2011, according to data from the Migration Policy Institute.
- 34% of Maine's foreign-born population age 25 and older had at least a bachelor's degree in 2011, compared to 28.2% of native-born persons age 25 and older.
- In Maine, 83.1% of children with immigrant parents were considered "English proficient" as of 2009, according to data from the Urban Institute.
- The English proficiency rate among Latino children in Maine was 94.1% as of 2009.


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The Princess Royal this morning met local people who, 40 years ago, rolled up their sleeves and helped restore the Caen Hill locks in Devizes.
The Princess arrived at the Town Bridge and boarded the Kenavon Venture canal boat for the short trip to the Wharf.
There she was introduced to the Caen Hill volunteers who are celebrating 40 years since they started their task which culminated in The Queen reopening the canal in 1990.
Peter Potter, of Victoria Road, said: "She was brilliant. She really knew her subject and talked about the canal restoration work in the Cotswolds.
"It was just like chatting with your auntie."
The princess also met members of Devizes Canoe club, volunteers from Crofton pumping station before handing over the Queen's Award for Voluntary Service to David Bruce, president of the Kennet and Avon Canal Trust.
Earlier the Princess Royal met the cream of Melksham's business community when she presented a local dairy exporter with its third royal award.
Princess Anne was visiting to present Coombe Castle with the Queen's Award for International Trade, and to officially open the firm's new headquarters.
She was shown around The Creamery, the company's new base at the former Falcon Interiors site in Western Way, visiting the production line, meeting employees and venturing into the four degree- chilled storage room.
She then presented the award to company chairman Glyn Woolley, who founded the firm in Corsham in 1980.
She said: "It is always a pleasure to be allowed to present the Queen's Award.
"Winning it once is impressive, but winning three is a real achievement, and something tells me you may be adding to your collection again in the future."
The Princess, whose son-in-law Mike Tindall plays for Gloucester Rugby, was presented with a selection of the firm's cheeses and creams, and a teddy bear in Bath Rugby colours, to which she joked: "I might have to take that off."
She was welcomed to The Creamery by town mayor Chris Petty, Duncan Hames MP, newly-appointed High Sheriff of Wiltshire Peter Addington and Sarah Troughton, the Lord Lieutenant of Wiltshire.
Mr Woolley said: "I feel very honoured that she came here. I wrote to her personally, and I'm very glad we were able to book a royal.
"We moved here last year, and we were able to combine two warehouses and all our staff into one place.
"There's plenty of space to expand here, and we're always looking to expand into new products, so who knows what we will do next?"
The firm exports British and Irish dairy products to 35 different countries, with many of its creams produced on-site in Melksham.
Mr Woolley said: "People trust British products, that's our biggest claim internationally.
"I only work three days a week now. I'm standing down soon so I'm focused on grooming a team who will be capable of taking the company into the next 30 years."


text:
It looks like NVIDIA are working on the next generation Kepler GPU. According to a social website profile, an intern at NVIDIA was working on a GK208 GPU during his time there between June and September, as can be seen in his/her experience profile. While it could just be a typo, it might well not be, since GPU development can begin years before release, especially the way GPU complexity has been rising in the last few years.
The intern's identity has not been revealed, to avoid embarrassment at possibly having unintentionally leaked sensitive information. There's not much more to add at this point except that this would be a low end next gen GPU. Anything more would be speculation, so we'll leave it at that.
Long story short, this information causes a lot of confusion. We were all convinced that the next flagship GPU of GeForce 700 Series would be GK110 or GK114. If the GK208 really exists then the whole line-up should consist of GK2xx silicones. This however doesn't stop us from speculating. We can assume that NVIDIA might somehow modify the GK110 for consumer and rename it to GK114, GK204, GK210 or GK214.
Our sources told us that the upcoming flagship of GeForce 700 series — GeForce GTX 780 — would feature between 1728 and 2112 CUDA cores. If we assume that the GK208 would be an entry-level GPU then it could likely power the GeForce GT 740 or GTX 750. If the 192 CUDA cores SMX architecture would not be changed, then GeForce GT 740/750 might be packed with 576 cores (3*192).
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text:
We are currently backing up User's home directories for a period of ONE Month after which, the disk space will be recycled.
We have deployed a large DLT (digital linear tape) tape backup system using Oracle StorEdge L1400 tape library. We have implemented a backup and retrieval strategy of user files located in each users home directory (i.e., all files/directories referenced by the $HOME environment variable).
Check out the Software Page for information about available packages.
HPCVL also has portable data storage solutions available for users that have very large data but limited bandwidth. Please see Portable Storage Sollutions for more information.


text:
|Product Description
The 2.25 in (57 mm) is compact and lightweight, with fast trim and release under high or low loads. Perfect for dinghies, scows, beachcats and iceboats, as well as small offshore racers and cruisers.
Delrin® ball bearings, sheave, and sideplates are UV stabilized with carbon black for maximum protection.
The wire version features Hardkote-anodized, Teflon® impregnated aluminum sheaves for strength and corrosion resistance.
Use for:
Main and jib sheets, Spinnaker halyards and Control lines.
Part Number: 004


text:
jubuttib wrote:Slower. They will slow down very quickly, and hence don't like to stay in the air and will fade earlier than proper MD2's.Sean40474 wrote:I just got 1/2 my order today, the PDs look nice. The C MD2 is domey has hell though. What are these domey ones flying like compared to the flatter ones? The dome on this is very much like my domey Z Buzzz.
So slower, less glidey and more LSS.
Boooo, sounds like a replica of my Buzzz, I'll see how the D looks when I get it.


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FORMER Swindon Town footballer Nile Ranger was ordered to pay more than £3,000 after kicking in the door to his own apartment block.
The striker was set to go on trial at Swindon Magistrates Court yesterday,for the incident which took place in April, but he changed his plea to guilty minutes before he was due to appear.
The 23-year-old said he was returning to Plaza 21 in the town centre after a night out when he tried to gain access via the carpark entrance.
He said the door was jammed, as it had been in the past, and so he kicked it in an attempt to force the door open.
On the third attempt it opened but only after breaking the door.
The judge ordered him to pay £2,345 in compensation for damaging the door.
As well as paying compensation, Ranger was also ordered to pay costs of £250, fined £500 and ordered to pay a victim surcharge of £50.
Sentencing the striker, who is now rumoured to be having trials at Leeds United, District Judge Simon Cooper said: "On returning home you attempted to gain access and gave the door a good thumping.
"You are a large man and did some damage. You were reckless. You accept that and I accept that. I can see you did not intend to deliberately cause damage.
"It was more from a bad temper and frustration but not with any intention to damage the door.
"This could have gone to full trial but I give you credit for your guilty plea."
CCTV footage emerged earlier this year showing the former Newcastle United player returning home with a woman in the early hours of the morning before he kicked the door three times to gain entrance.
Before the sentence was given Tom Roberts, defending, read a statement on behalf of the footballer.
"I plead guilty to the charge of criminal damage," he read.
"I accept I kicked the door three times to gain access. The door was unreliable and I had had difficulty opening it before."
In May, Ranger also pleaded guilty to causing criminal damage to a taxi in Liverpool after a cabbie refused to wait for him.
The player spent one year at Swindon Town but parted company by mutual consent after the club tired of his off-the-field antics.
Ranger did not comment after yesterday's sentencing.