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Journal of RNA and Genomics
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- Journal of RNA and Genomics (2005) Review Article
Vector-based siRNA delivery strategies for high-throughput screening of novel target genes
Meihong Chen1, Quan Du2, Hong-Yan Zhang2, Claes Wahlestedt2,3 and Zicai Liang2*
1Chinese Human Genome Center North, Beijing, 100176, China and Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), National Laboratory of Medical Molecular Biology, Beijing, 100005, P. R. China.
2Center for Genomics and Bioinformatics, Karolinska Institutet, 17177, Stockholm, Sweden.
3Department of Drug Discovery, Scripps Florida, Jupiter, FL 33458, USA
Corresponding Author: Zicai Liang, Email: [email protected], Tel: +46 8 5248 6371, Fax: +46 8 323950
Received 10 June 2005; Revised 16 June 2005; Accepted 19 June 2005, Available online 27 July 2005; Published 12 August 2005
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Application of siRNA in high-throughput fashion is still in its early phase although the principle has been established for three years. In this review, we outline the different vector-based siRNA delivery platforms as well as resources that are becoming available for high-throughput applications, and some initial outcomes of vector siRNA high-throughput screening efforts using vector encoded siRNA. It is expected that further improvement of the siRNA technology and availability of the siRNA resources will help to materialize the potential of siRNA for functional genomics and drug target validation.
siRNA, RNAi, vectors, gene silencing, siRNA delivery
RNA interference (RNAi) achieved by double-stranded RNA (dsRNA) mediated sequence-specific breakdown of the mRNA (Fire et al, 1998; Montgomery and Fire, 1998; Elbashir et al, 2001a). It is an evolutionarily conserved phenomenon initially characterized in C. elegans and Drosophila, but also occurs in plants, fungi and mammals (Boshare and Labouesse, 2000). Mechanistic studies have indicated that the effector molecules of the RNAi process is not the long dsRNA itself, but a family of short doublestranded RNA fragments (19-21 base pairs in length) derived through cleavage of the long dsRNA by RNase III enzyme(s) (Elbashir et al, 2001b). The short doublestranded RNA fragments generated as such, as well as through chemical synthesis or vector-based expression, are collectively referred to as small interfering RNA (siRNA). The long dsRNA cannot be used in mammalian cells for gene silencing due to the fact that introduction of long dsRNA elicits a strong antiviral response that obscures any gene-specific silencing effect. Much of this response is caused by activation of the dsRNA-dependent protein kinase PKR, which phosphorylates and inactivates the translation initiation factor eIF2a (Proud, 1995). Introduction of siRNA into mammalian cells was found not to elicit the antiviral response to such an extreme level, but still can result in efficient gene knockdown (Sledz et al, 2003; Elbashir et al, 2001b). This makes siRNA widely applicable in various mammalian species. The mechanism and biological implications of RNAi are still not fully understood.
Methods for generating siRNAs
Short siRNA molecules can be prepared by direct chemical synthesis or transcription driven by RNA polymerase promoters. In term of high-throughput applications, vector- based strategies are favoured because such strategies enjoy the advantages of much lower cost and ability to regenerate. A synthetic siRNA library could be expensive for most researchers. The only large-scale synthetic siRNA library that was produced so far is the genome-wide siRNA collection made for Novartis by Qiagen (Hilden, Germany) and Dhamarcon (Lafayette, USA). Transfection efficiency of siRNA into cells depends on cell types and the RNAi effect of synthetic siRNA only sustain for a period of time. The advantage of the vector-based siRNA is the capability of removing those cells that are not transfected with the plasmids by selecting the transfected cells with antibiotic resistant genes. Virus vectors also enable the delivery of siRNA expression cassettes into cells with higher transfection efficiency, and in case of lentivirus and retrovirus, it is easy to make stable knockdown cells by integration into the genome. If one wants to use siRNA expression vectors as a combinatorial library, the viral vectors would be most suitable. In the current review, we will focus on the vector-based siRNA libraries and the methods to produce them.
siRNA production by pol III promoters
The use of pol III promoters in vectors for encoding siRNA was pioneered by a number of groups in early 2002 (Brummelkamp et al, 2002; Miyagishi and Taira, 2002; Sui et al, 2002; Xia et al, 2002; and Yu et al, 2002). The vectors contain a single pol III promoter followed by a segment of DNA that encodes a short hairpin RNA (shRNA) (Figure 1A). The shRNA closely resembles the structure of microRNAs and will be integrated into the RISC complex in the same way as the normal siRNA does. The backbones of these constructs were initially plasmids, but the grafting the shRNA expressing cassettes into different viral vectors such as lentivirus, retrovirus and even adenovirus also explored successfully to facilitate the transfection of cells that were hard to penetrate, such as primary cells and suspension cells (Li et al, 2003; Hosono et al, 2004; Devroe and Silver, 2004)
Another strategy of encoding siRNA that has gain more popularity is a vector that contains dual pol III promoters arranged in a convergent manner that drive the expression of two short complementary RNA strand from a single 19 bp DNA region (Figure 1B) (Chen et al, 2005; Zheng et al 2004). While being as effective as the shRNA encoding vectors, these dual-promoter (GeneBuster) vectors are first of all more robust for cloning both because of the lack of secondary structure in the siRNA coding region, and much lower level of DNA synthesis errors due to the shorter sequence, and they have tremendous advantages in constructing large scale siRNA libraries in a combinatorial way, as will be discussed below.
A third method is a technique using two tandem pol III promoters to drive the expression of siRNA from separate 19 mer DNA fragments (Figure 1C) (Lee et al, 2002). This method might not be widely used due to the fact that construction of such a plasmid is considerably more cumbersome than the other two methods. This method as well as the shRNA method, however, offers the possibility to integrate mismatches into the resulting siRNA, thus possibly enhance the efficacy of the siRNA or increase the frequency of effective siRNA.
H1 and U6 promoters from human and mouse are the most widely used pol III promoters in the siRNA vector construction. Other promoters that have been used include promoters of tRNAVal and tRNAMet (Oshima et al, 2003; Boden et al, 2003). All these pol III promoters are active in a large variety of cells, and appear to perform similarly in different species. This makes it very easy to use the same vector in different experimental systems. One interesting feature of the pol III promoters is that they can be easily transformed into inducible promoters (van de Wetering et al, 2003). This feature is very important for some functional studies and screening.
In addition to plasmid-based systems, PCR-derived siRNA expression cassettes based on the single-promoter system have been shown to efficiently suppress gene activities in transfected cells (Castanotto et al, 2002).
siRNA production by other promoters
Other promoters that have been used for siRNA production include T7 promoter and CMV promoter (Xia et al, 2002; Holle et al, 2004). Since T7 promoter does not work in mammalian cells, strategies employing T7 or similar promoters normally can only support siRNA production in test tubes, but not within cells. People have overcome this shortcoming by either pre-load the promoter with CMV promoter is a much stronger promoter compared to other pol III promoters, and this can allow more siRNA molecules to be transcribed from a given amount of DNA vector. But since CMV promoter is an RNA polymerase II (pol II) promoter, the resulting transcripts are normally capped at the 5'-end and tailed at the 3'-end with a long poly (A) sequence. It appears that such modifications are well tolerated as shown in the efficacy assays. Due to the potentially higher degree of flexibility in enabling different spatial and temporal expression patterns, these types of vector-based siRNA strategies might be much more useful for in vivo research purposes and for siRNA based genetherapy at a later stage although pol III based vectors seem to be able to satisfy most of the gene knock-down needs in cultured cells.
Construction of siRNA libraries
One of the critical initial steps in the high throughput siRNA application is the formation of a physical siRNA library, which many people also refer to as a siRNA library.
To generate a siRNA library, one critical factor to consider is the design of siRNA sequence. Since the first guideline for siRNA design was published by Tuschl and colleagues (Elbashir et al, 2001b) several studies of currently available information for effective and non-effective siRNA have been carried out, and these have resulted in different siRNA design tools that based on only partially overlapping criteria (Henschel et al, 2004; Wang and Mu, 2004; Chalk et al, 2005). SiRNA vendors have also made their own siRNA design software available on the web.
One common drawback of all currently existing siRNA design software is that they are all based on limited data sets about siRNA efficacy. It is anticipated that, with the development of high throughput siRNA construction and validation methods, algorithms based on randomly chosen siRNA will emerge to provide better predictions for functional siRNA.
Although a siRNA library can be constructed by chemical synthesis, as done by Qiagen for Novartis and more recently by Dharmacon, most of the siRNA libraries covering more than a couple of thousand of genes are done by vector-based approaches. This is because, i) a vector-based siRNA library can be regenerated with minimal effort, ii) the constructions can be carried out in a standard molecular biology laboratory, and iii) a vector-based siRNA library can be much more affordable than a chemically synthesized library.
Figure 1. Diagrams of three general ways of encoding siRNA in a plasmid or viral vector. The same principles apply also in PCR cassettes that derive from these vectors. A, a hairpin-like siRNA (shRNA) is generated by transcription from a segment of DNA (50-60 bp in length) driven by a single promoter for pol III RNA polymerases. B, expression of a siRNA from a single 19-mer DNA fragment through transcription driven two "opposing" promoters for Pol III RNA polymerases. C, expression of a siRNA from a two separate 19-mer DNA fragments through transcription driven two "tandem" promoters for Pol III RNA polymerases.
Three large-scale siRNA libraries have recently been constructed by academic researchers (Paddison et al, 2004; Berns et al 2004, Michiels et al, 2002) and Galapagos has generated the first large scale adenovirus based siRNA library in the industrial sector. Multiple efforts have been initiated since then by commercial companies, and notably among them, Genordia AB, a Sweden-based company will most likely be able to deliver the first siRNA library that covers a whole mammalian genome in 2005.`
Hannon and colleagues have constructed a large-scale siRNA library targeting a selected panel of human and mouse genes using a hairpin siRNA encoding cassette (Berns et al, 2004). The siRNA-encoding cassette was first cloned into a pSHAG-Magic donor vector. Although in most siRNA applications people has chosen to use 19- 21mer siRNA, this particular siRNA library encoded siRNA expression unit that contained 29 nucleotides, simple loop structure and U6 promoter. It was found that 29- nucleotide hairpins were more effective than shorter hairpin siRNA in this study. Since the efficacy of short siRNA is well established, this interesting finding might need more independent confirmation before being adopted as a guideline for siRNA libraries construction. Also concerns have been raised for possibly higher level of off-target hits as well as induction of antiviral responses. Totally 28,659 shRNAs targeting 9,610 human genes and 9,119 shRNAs targeting 5,563 mouse genes were included in the Hannon siRNA library. Some characteristics of this library include: 1) all clones were sequence verified. The extensive sequence verification was warranted because 25–75% of cloned shRNAs were found to contain significant mutations that arose during chemical synthesis. 2) Only coding sequences were targeted, and each shRNA was chosen such that it contained not more than 26 bp matches to any other gene. It should be cautioned that recent studies have demonstrated that for processed siRNA sequence identity up to 16-17 might already allow the siRNA to have offtarget effects. 3) Where possible, shRNAs were designed to have sequence identity to the mouse orthologue of the targeted gene. In terms of gene family representation, this library covers about 85% of all known kinases and phosphatases by three or more siRNAs per gene. Most other functional classes contain between 30–60% coverage with three or more hairpins, and 80% of genes by at least one sequence-verified hairpin. A unique 60-nuclotide DNA bar code has been integrated to each vector to allow identification of shRNAs in populations of virally transduced cells through hybridization methods.
The human RNAi library (the 'NKi library') constructed by Bernards and colleagues contains 23,742 siRNA vectors that cover 7,914 human genes (Michiels et al, 2002). Gene within this collection include components of major cellular pathways, such as the cell cycle, transcription regulation, stress signaling, signal transduction and important biological processes such as biosynthesis, proteolysis and metabolism. In addition, genes implicated in cancer and other diseases are included in the library. The siRNA encoding DNA fragments were cloned in a high-throughput fashion into pRetroSuper (pRS), a previously reported retroviral vector containing the shRNA expression cassette. This is one of the first large scale siRNA collection in the academic sector and in the validation of this library, researchers have achieved on average 70% expression inhibition for approximately 70% of the genes in the library using a pool of three knockdown vectors against a single gene. Compared to siRNA library made in plasmid vectors, siRNA librarys constructed in viral vectors has to be packaged and mass produced before being used in any knockdown experiments. These might comprise a major limitation in the application of viral vector based siRNA libraries in very large-scale screening.
Schultz and his colleagues have developed a PCR based system to generate siRNA for about 8000 genes at 2- siRNA/gene coverage using an opposing promoter approach. The siRNA has been used to discover novel members of the NF-kB pathway (Zheng et al, 2004). A singlestep PCR protocol was used to produce siRNA expression cassettes in a high-throughput fashion and showed that these siRNA expression cassettes can also specifically and efficiently suppress the expression of both transfected and endogenous genes. This single-step PCR approach is efficient and cost-effective and makes high-throughput production of siRNA expression cassette libraries for genome- wide functional gene annotation practical. The arrayed library was used to screen for genes involved in the NF-B signalling pathway that controls many diverse cellular processes including growth, development, inflammation, immune response, apoptosis, and oncogenesis.
Notably, one of the first viral based siRNA libraries was generated by an industrial player, Galapagos Genomics. This adenoviral siRNA library contains knock-down reagents for over 4,900 human druggable transcripts, including G-protein coupled receptors (GPCRs), ion channels, nuclear hormone receptors, kinases, phosphodiesterases and other druggable transcripts. Three adenoviral vector encoded siRNAs were designed for each gene. This threefold redundancy in siRNA sequences gives a 90% probability that the mRNA for any given gene will be knockeddown by at least 75%. Such a loss-of-function gene collection provides an ideal complement to the arrayed adenoviral gain-of-function libraries from the same source. Other characteristics of this siRNA library include: 1) the siRNA expression cassette is based on the human U6 promoter, a strong, ubiquitously active promoter that lacks essential promoter elements within the transcribed region. 2) The U6 promoter-based expression cassette was cloned in an adenoviral adapter plasmid at the position of the E1 region. The adenoviral vectors were based on adenovirus serotype 5 with deleted E1 and E2A regions. 3) The viral vectors were found to be able to express processed hairpin RNAs for at least 10 d. 4) The adenoviral vectors used have a broad tropism and very efficiently infect many different primary cell types including primary keratinocytes and primary synoviocytes, two cell types that are notoriously difficult to transfect. The use of siRNA tools in primary cells has been hampered by inefficient transfection protocols. Advantages in the transfection aspect are of critical importance for some applications, and thus counter-balance the burden imposed by the cumbersome viral handling.
siRNA libraries with degenerated sequences
In a conventional siRNA application, a target gene is chosen first, and then siRNA sequences are designed against the transcript of this gene. In this approach, the choice of gene, and choice of targeting sites are inevitably biased due to the limited number of genes and sites that can be included in a particular study. The bias also arise inevitably from the fact that we have just started to get a relatively complete picture of the part of the mouse transcriptome that has a poly (A) tail, and knowing little about transcripts that do not have a such a tail. The development of siRNA expressing libraries that encode substantially all permutations of 19-mer siRNA thus become an appealing approach to allow the use of it as a single affordable tool for un-biased genome-wide screening.
Towards this direction, siRNA libraries have been synthesized by integration of 19-mer fully randomized DNA sequences into different siRNA encoding vectors (Chen et al, 2005). Such vectors should have theoretical complexity of 2.75x1011 in order to encode all the permutations of siRNA. This is a complexity that, albeit being reachable, will impose significant burden on the manipulation of the library and use of it for screening. Lucky or not, the siRNA was actually found to be tolerant to mismatches towards its terminus. This makes it reasonable to assume that a library will about 1 x 109 complexity will cover all possible siRNA. Such a complexity then comes in a range that is practical for plasmid library construction and maintenance. The library was used for phenotype-driven screening using cell proliferation as a model, and multiple siRNA that can induce significant enhancement of cell growth were identified.
Another alternative approach is to generate semi-random siRNA library from an mRNA source. In this case, a specific mRNA population is converted into double stranded cDNA, which will be cut into short DNA fragments that can then inserted into the vectors for encoding siRNA. The advantage of this type of libraries is that it will only have a complexity of in the neighborhood of 106 but already be able to cover a substantial part of the genome. The limiting aspect of the approach is that a library made in this way will only be useful for the same organism, or even only for the same cell type, whereas other fully randomized libraries can be used in all cell types and all relevant organisms. There have been several reports (Luo et al, 2004; Sen et al, 2004; Shirane et al, 2004) for the construction of such semi-random siRNA libraries and one of these is briefly described here as an example (Sen et al, 2004). In this method, mRNA is converted into double-stranded cDNA mixture that was further cleaved with a mixture of five restriction endonucleases, each of which leaves a 5'-CG overhang but has a different 4-bp recognition sequence. This combination covers 6 of 256 possible 4-bp sequences, resulting in one cleavage every 43 bp. These fragmented cDNAs are ligated to a hairpin linker with a complementary 5'-CG overhang. This hairpin has a recognition site for MmeI, a type II restriction endonuclease that cleaves 20–21 bp away from its recognition sequence and leaves a two-base 5' overhang. A hairpin linker is then ligated to either the 5' or 3' end of the double-stranded cDNA fragment. Next an "extension linker" comprised of three different oligonucleotides is ligated to the cDNA-extended hairpin. As a result the 5' and 3' ends of the extended hairpin become ligated to the extension "sense" oligonucleo tide (green) and the extension "antisense" oligonucleotide respectively. The extension priming oligonucleotide serves as primer for DNA elongation by the highly processive Bst DNA polymerase large fragment, which lacks a 5'–3' exonuclease domain. This reaction converts the hairpin into a dsDNA that encodes a hairpin RNA. The process has been used to construct a siRNA library from a mouse embryo cDNA library. This resulted in three million independent clones. Twenty-seven clones were chosen at random for sequencing, all of which revealed matches against known mouse genes. Five random clones were used to generate stable NIH 3T3 cell lines. Northern blotting with probes corresponding to their mRNA targets demonstrated that each of the cDNA-derived inserts produced the expected processed 21-base siRNAs and these clones caused specific reductions in their corresponding target mRNA.
Functional screening using different siRNA libraries
Genome-wide RNAi screens were explored in C. elegans using libraries of in vitro-transcribed long dsRNAs or simply the bacteria that express dsRNA. For example, an RNAi feeding library consisting of 16,757 bacterial clones covering majority of the worm genome was constructed (Fraser et al, 2000; Kamath et al, 2003). Upon feeding the worms, these clones generated transient loss-of-function phenotypes for many genes by inactivating the target genes via RNAi. Genome-wide RNAi approaches have been used successfully for phenotype-based screens also in Drosophila melanogaster. In part, these successes derived from the availability of convenient and inexpensive methods for producing and introducing dsRNA. To date, the use of RNAi libraries in mammalian systems is becoming well accepted but most of the work has been limited to specific protein families because of technical and practical issues associated with generating large synthetic siRNA and/or vector-based short hairpin RNA (shRNA) expression libraries, but the situation is rapidly improving with the technology development mentioned above. Screening schemes using large-scale siRNA libraries are understandably diverse due to differences in aims, vector systems, and read-out methods. The five case studies presented below could give us a hint about how such libraries can be deployed in HTS.
Retroviral vectors can be used to transfect a large variety of cell types, and its short-coming on the handling side can be well balanced by using it in a combinatorial manner, e.g., in libraries. Work done by Berns et al (2004) has provided a typical example of such applications in a medium scale. In this study, retroviral vectors were generated in pools and then the pools were used to transfect BJ-TERTtsLT cells at 32 °C. After two days' incubation at 32 °C, the incubation temperature was increased to 39 °C for proliferating colony screening. In the first-round screening, six pools of infected cells were found able to form colonies at 39°C. These shRNA inserts were then re-cloned from these colonies, and their identity was established by DNA sequence analysis. Only those shRNA inserts that were presented in multiple independently derived colonies were further analyzed in the second-round screening in BJT ERT-tsLT fibroblast. Using this approach, shRNAs against six genes were identified that could suppress the temperature-shift-induced proliferation arrest in the BJ BJT ERT-tsLT cells. Remarkably one identified gene was p53, which underscores the quality of the NKi library. Knockdown of other five identified genes also mediated escape from proliferation arrest in BJ-TERT-tsLT cells when tested in the absence of retroviral vector, suggesting that inhibition of these genes allowed bypass of the p53 response. In screening as such, it would always be a concern whether the change in read-out is a consequence of on-target hit or off-target hits. In this case, it was found that two of the three shRNAs targeting a single gene could mediate escape from growth arrest for three of the five newly identified genes. This could be considered as a confirmation that the effects of the siRNAs were 'on-target'. For genes that only have a single siRNA hit, normally additional siRNA need to be designed to confirm any phenotypical changes observed with the initial siRNA clone.
In a similar screening, 6712 shRNA expression targeting 4873 genes were used to discover novel members of proteasome. The 26S proteasome is the major non-lysosomal protease in eukaryotic cells. To search the library for shRNAs that compromise proteasome function, a reporter assay was used in which a fluorescent protein (Zoanthus green fluorescent, ZsGreen) was coupled to a well characterized degradation signal, the mouse ornithine decarboxylase (MODC) gene. Another plasmid encoded Discosoma red fluorescent protein (DsRed) was used as a normalization control for transfection. Individual transfection of 6,712 shRNAs revealed approximately 100 RNAi constructs that increased the accumulation of ZsGreen– MODC, of which 22 corresponded to 15 known proteasome subunits.
As mentioned above, a siRNA expression cassette library targeting 8,000 human genes from the public UniGene library were generated in Schultz lab in Scripps Institute in a vector containing dual pol III promoters, with two targeting sequences per gene Zheng et al, 2004). To screen for regulators of NF-kB transcriptional activation by TNFa, the pNF-B-Luc reporter plasmid was cotransfected with individual siRNA expression cassettes into HEK293T cells. Of 94 genes identified in the initial screening, 20 hits were further assessed by introducing additional siRNAs, and 17 of them seemed to be genuine on-target hits. Among them, eight are known to be involved in the NFkB signalling. Of the remaining genes, some might be involved in cellular processes that indirectly affect the NF-B pathway, whereas others might have previously unrecognized roles in the NF-B signalling pathway.
All screening work mentioned above were done in microtitre plates, which will remain a major screening platform in the future. Cell microarrays were created to provide much higher throughput than microtitre plates for siRNA based screening. Cell microarray was first described by Ziauddin and Sabatini (2001) who demonstrated that cells grown on a glass substrate could take up DNA–lipid complexes that had been deposited on the slide before cells were plated. Cells became transfected in situ, with defined spots of transfected cells localized over the printed DNAs. Cell microarrays represent a novel alternative to classical approaches to phenotype-based assays in mammalian cells, at least for some of the easy-to-transfect cells such as HEK 293T, IMR90/E1A, NIH 3T3, and HeLa cells. In this screening format, siRNA (plasmids or PCR cassettes) are spotted onto glass slides together with transfection reagents (normally with gelatin too for immobilization purpose). The vector based siRNA reagents appear to be stable for months on such chips. When cells are overlaid on top of a spot on the slides, corresponding siRNA vector will be taken into the cells so that siRNA will be expressed, and the effect of the siRNA can be read out in high throughput according to the phenotypes or the reporter systems used. The initial proof-of principle was done by siRNAs corresponding to the enhanced Green Fluorescent Protein (GFP) gene (eGFP) on microscope slides, and plated HeLa cells permanently expressing a destabilized version of eGFP on top of the slides. The result showed the cellular uptake, "reverse transfection", of rhodamine-tagged siRNA (rh-eGFP), and silencing of GFP expression in cells. An experimental comparison was provided by carrying out, side by side, the array-based and microtitre plate screening of siRNA that can perturb proteosome functions. The result showed that the array method does work in screening for siRNA that can manipulate endogenous genes, and likely to provide data that is even better than the classic microtitre plate based screening process. Advantage of the method is that large number of siRNA can be rapidly screening through a given phenotype or reporter system. The limitation of the method is, in additional to the need of complex instrumentation, that many phenotypes can't be fit into such an array- based read-out format.
RNAi provides a perfect example of how a natural occurring process can be "hijacked" by researchers and used as a research tool. The penetration speed of this technology into the labs in both academic and industrial settings has been astonishing even though the mechanism of RNAi has not been fully understood. In this reveal, we tried to focus on the development and application of vector based siRNA libraries due to that fact that although synthetic siRNA has become much more affordable now than two years ago due to stiff competition and improvement in the throughput of RNA synthesis, vector-based siRNA will still be the reagent of choice for large scale siRNA based screening in most academic institutions and companies. It is worth to stress that synthetic siRNA has also certain advantages over vector-based siRNA approaches in dosing, integration of modified nucleotides, and delivery, in just name a few aspects for example.
It is anticipated that plasmid libraries that have a genome- wide coverage will become available in 2005 for screening using both transient transfection as well as stable transfections. Inducible promoters will be integrated into some of the libraries for special screening need. While lentivirus and other viral vectors provide obvious advantages in the transfection of hard-totransfect cells, they are bound to be significantly cumbersome to work with in a high throughput screening. It is more likely that they will serve as second line of verification tools instead of being used as the first line screening tools in many occasions other than combinatorial screening.
The authors wish to acknowledge Pfizer Inc and the Chinese High Tech Research and Development Program (863) for support.
Authors Hong-Yan Zhang, Claes Wahlestedt and Zicai Liang are founders of a reagent company, Genordia AB, specializing in the development, production and sales of RNAi-related products.
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I am glad the kid is gone.
Worst episode of Camp yet and there is no chance for the only relationship that looked interesting.
She's not comitting a felony or geting married. Right now, for us, that's a win.
On a plus side Toby doesn't have chlamydia.
They really should have gone with the twin story, who starts a revenge because of getting out of college? There are so many other things you can do with your life. And who decides this would be a good storyline? The idea of Mona being A was more intriguing. Cece (I am assuming and hoping she's not the main A shouldn't even be on the A team, she has no business in Rosewood anymore she is much older than the girls should cope with her own mistakes with some maturity.


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Kofola: The Czech Coca-Cola
The origins and continuing success of one of the Czech Republic's enduring brands
Ryan Scott
Czech Companies, Food & Drink
The soft-drink Kofola is one of the most famous Czech brands. Its stylized logo is as much a part of the local commercial landscape as Škoda, Baťa, or Pilsner Urquell. Yet all these companies started before communism, even before Czechoslovakia was formed. Kofola, on the other hand, emerged during the former regime and despite some setbacks has remained one of the country's best loved products.
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From Anti-Imperialist Drink to Soft-Drink Empire
In 1959, chemist Zdeněk Blažek was tasked by the company Galena with finding a way to use surplus caffeine and develop an alternative to 'West imperialistic Coca-Cola'. The fizzy symbol of capitalism was certainly available during the communist period, but it was expensive and available from Tuzex or restaurants. Blažek's product was the syrup Kofo, made from herbal ingredients, fruit extracts and caffeine. Kofo was added to carbonated water, creating Kofola, which sold for half the price of foreign colas and was more widely distributed. This undoubtedly gave the drink a huge advantage in the once tightly controlled market.
Despite the lack of a market economy, Kofola engaged in some simple marketing during communism, such as this New Year's Greeting card.
The image of an attractive young woman suggests that the ideas about product promotion the company had back in the seventies were not too far away from the West at the time – i.e. youth and beauty sells, even if the women looks somewhat melancholy.
The transition to an open economy brought greater competition from foreign brands, namely in the reduction of their prices. This change, along with the euphoria of shedding communism, the association of Coca Cola and Pepsi with the West, the allure of these formerly restricted products, and finally Kofola's association with the bad old days all contributed to a decline in Kofola's sales.
The slump was short lived. In 2002, Santa, a drinks company owned by Greek expatriate Kostas Samaras, acquired both the brand name and the recipe from American pharmaceutical firm IVAX for 215 million CZK. Today, Kofola is one of the biggest drink producers in central Europe. It is certainly the biggest domestic soft drink maker, controlling 32% of the market, which places it in second place behind Coca Cola which has about 35%. Pepsi's share is only about half that number. All from a product considered commercially dead two decades ago.
Also read: The 10 best things to do in Prague this week: January 13–19, 2020
The Path to Resurrection
The story of Santa is as interesting as the brand they own. Kostas Samaras left his native Greece as an 8 year-old boy in 1948 during the country's civil war, which raged from 1946 to 1949. He was separated from his parents and placed in a Czechoslovakian orphanage.
Studying agricultural engineering at university, Samaras saw little future in socialist agriculture techniques after the revolution, and switched to running a company which imported goods from Greece. The venture was not a success and he nearly lost all of the property which he'd put into the company.
In 1993 Samaras, now a father, bought a soda drink plant in Krnova. His son, Jannis, the current head of the company, left university to work with his father. Originally, the plant made and bottled a lemon-flavored syrup, later juices and soda. With the acquisition of Kofola, the company has become one of the leading Czech brands.
Various media accounts portray Jannis as a humble and reserved man. Stories describe that he doesn't wear a suit to work, and when he was named the businessperson of the year in February he had to borrow a tuxedo for the award ceremony. There is a sense that this down-to-earth image is part of Kofola's appeal.
The Recipe for Success
The recipe for the drink remains a secret, with only five people in the company knowing the drink's ingredients. Václav Klaus also knows. It's not some presidential privilege. The company granted him knowledge as a seventieth birthday present.
Despite being a secret, key ingredients are known, even if their proportions aren't. Apart from sugar, the drink contains a mix of spices, citrus extracts and extracts from sweet woods and the leaves of blackberries and raspberries. The company claims that Kofola has less sugar than other colas and no phosphoric acid. But it does contain more caffeine.
Also read: Prague's Old Town Market renovations are now underway, but will cost more than originally projected
One apparent change in the mix has been a reduction in saponins. One article alleges that the quantity of these foam producing agents was so high that a bottle of Kofola left in the sun could explode.
Taste of Love / A New Recipe for Success / Love for Sale
An added ingredient to the company's rebirth is its modern marketing campaigns, which paradoxically rely on nostalgia and tradition. Using the slogan Když ji miluješ, není co řešit (When you love her, nothing else matters), the company has rebranded itself as a quirky somewhat irreverent product, producing what have been some very well received ads.
The appeal to days gone by was the result of market research, as explained by former chairman of the board René Sommer. He said in the interview at the link that people lived through many terrible things, but they recall some positive events too, and one of those might be drinking Kofola as a child.
This ad, shot for the companies fiftieth anniversary, taps into a certain nostalgia for the sixties. It shows how a chemist is trying to discover the literal chemistry of love but discovers Kofola instead.
Another ad focused more directly on the theme of young love.
And sometimes the company isn't afraid to venture into taboo topics.
Some ads have taken a different approach. The Christmas ad, which was very well received, and according to Sommer, viewed by people of a sign that Christmas was coming, makes no reference to the drink until the end.
It is based on a Czech saying that if you fast over Christmas Eve, you will see the golden pig at dinner time, which is what the father is explaining to his daughter. As he cuts down the tree she asks him what the pig looks like, eventually announcing that she can already see him.
Also read: Celebrate the Lunar New Year with a Taiwanese feast in Prague
Broadening Tastes
Like other soft drink producers, Kofola has responded to changing and expanding palettes of consumers by introducing new varieties. The other types include Kofola bez cukru, which is sugar-free, Kofola Citrus, Kofola Extra Bylinková which includes gentian, dandylion and peppermint in addition to the regular herbs, and a sour cherry Kofola called Višnová.
The company has also started to sell the drink in cans to keep up with demand, and have expanded into other drinks such as the mineral water Rajec and energy drink Semtex and Erektus.
The proven success of Kofola, along with changing tastes and an interest in local products, could be opening the way for other local soft drinks, known as limonáda or limča, irrespective of whether they are lemon flavored.
One of the most commonly available is malinovka which as you know no doubt guessed is a fizzy raspberry drink. After some decrease in sales, interest in soft drinks has started to rise again. Perhaps the increase is the stricter rules concerning drunk driving mixed with the retro appeal of these products. However, it seems unlikely that the soft drink market is going to make a dent in Czech beer consumption, which is still the highest per capita in the world at about 150 liters per person annually.
What do you think of Kofola?
Farewell Fake Rum? A Short History of the Endangered Czech Spirit
Cocktail Emporium with Prague-Themed Drinks Opens on Wenceslas Square
Prague Named among Top 10 Vegan-Friendly Capitals In 2017
Czech Republic Will Continue to Fight Inferior Food Quality
Take a Look Inside Prague's New Temple For Meat Lovers
The 10 best things to do with kids this week in Prague
Czech-filmed comedy Jojo Rabbit gets six Oscar nominations, animated short Dcera earns one
Prague and Taipei make it official as mayors sign partnership agreement between their cities
Shanghai suspends all official contact with Prague over its Taipei partnership agreement
Prague 1 banning electric scooters in pedestrian zones
Prague Zoo raising money to help Australian wildlife
Jan Palach memorial to commemorate 51st anniversary of martyr's death
Czech Pirate Party to push for legalization of cannabis as well as LSD, ecstasy, and mushrooms
Opatov metro area to be renovated with new square and buildings
Celebrate the Lunar New Year with a Taiwanese feast in Prague
For the first time ever the Czech-Taiwanese Association will host a public Lunar New Year's Eve dinner
The 10 best things to do in Prague this week: January 20–26, 2020
Kick (and stomp and roll) away the winter blues with a week full of out-loud fun in the Czech capital
Prague's Old Town Market renovations are now underway, but will cost more than originally projected
The Old Town Market has fallen into disrepair but will reopen as center for cuisine and culture
Kicking off in Prague this week: a citywide food festival, films galore, world-class art exhibits, and a strapping new show
The 10 best things to do in Prague this week: January 6–12, 2020
Post-holidays it's time to practice a little hygge in Prague: snuggle up with coffee, board games, knitting, and Scandinavian film
50 last-minute tips for spending a dazzling New Year's Eve 2020 in Prague
From gala events to laid-back fun, these are some of the best places to ring in the year 2020 in the Czech capital
Why do Czechs eat carp for Christmas?
A complete history of this fishiest of festive Czech meals from the Middle Ages to today's Christmas fry up with a potato-salad side
The 5 best activities for families in Prague this week: December 23–29
With the kids off school this week here are some tips for activities leading up to and after the Christmas holiday
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Eating & Food:Health & Medical - CbwChina News & HeadLines.
Making homemade meringue cookies is easy and saves money. They are a great basis for desserts and do not need refrigeration.
This easy cake mix cake is made with raspberry jam, a chocolate cake mix, Chambord or milk, and other ingredients. The chocolate raspberry cake recipe is baked in a Bundt cake pan.
Six delicious Swiss chard recipes, including a crustless chard quiche, creamed Swiss chard, Swiss chard with bacon, and more. Try one of these flavorful Swiss chard recipes today!
Arancini di riso are Sicilian street food, fried rice croquettes with fillings (often ground meat). The idea sounds simple, but they're one of the finest snacks around, and are also an excellent party food.
Here is a tip for packing-up cupcakes for lunchboxes, picnic baskets or the freezer.
Thyme is easily dried at home. Here's how.
Helpful tips on how to select, store, and dry your own thyme at home.
Crockpot apricot beverage recipe with apricot nectar, spices, orange juice, and lemon juice.
We are the innovators of culinary teambuilding programs.In our kitchen, we will challenge and improve your team like productivity boost, team building, team motivation, culinary team building, team building actvities,teams,sales team. We are located in NewYork, USA.
If you like peach or apricot pie, try this easy papaya pie for a touch of the tropics.
In the past it was not uncommon to find meat in every other dish prepared. But, things are changing now and health concerns being one major reason for such change. Animals are chemically injected to beef up and such chemicals are affecting their health and in turn ours.
If you are looking at the option of redefining the special occasion in your life, you can always consider opting for the Photo Cakes in Delhi.


Home > Podcast > Increase Your Impact by 95%!
Increase Your Impact by 95%!
While the 5% grantmaking payout certainly furthers your foundation's philanthropic goals, what are you doing with the other 95%? Increasingly, private foundations are exploring the benefits of impact investing, aligning their investment portfolio with their missions to improve their impact. In this podcast, Kristin Hull shares her passion for impact investing, which inspired her to allocate 100% of her family foundation's assets to mission-related investments. A signatory of the Divest Invest Philanthropy Pledge, Kristin also offers advice on how next-generation trustees can introduce impact investing to their family foundations.
Kristin Hull is an educator, entrepreneur, philanthropist, and advocate for impact investing particularly among family foundations. Kristin began her career as a bilingual educator. Interested in innovative solutions for social change, she entered the field of impact investing while serving as a board member for various non-profit organizations. After the sale of her family's business, she helped align the entirety of her family foundation's investments with its mission and then started the Nia Community Fund, a private foundation concentrating on social equity and environmental sustainability in Oakland, California.


Telling the story will talk about how to portray our vision and tell the story rather than concentrating on pure aesthetic and polish. The story not the frame is the king.
Day 1 morning: will cover equipment choice, the importance of video and why it is different to the still image, researching a story, planning the shoot, shot lists and gear preparation. Angles, framing and format will be discussed as will audio.
afternoon: practical session around Winchester with tutor and then debriefing, Q&A and be given a task to prepare for the 2nd day.
Day 2 morning session: review and group discussion/critique of participants videos. What could have been done better and how.
afternoon session: editing techniques for impact and flow. Group shoot with tutor demonstration and questions from students.
This workshop assumes no prior knowledge of camera video work, but does assume a knowledge of the camera you are using.
Participants are expected to attend both sessions and to make the time to prepare a short video between Day 1 and Day 2.
Documentation will be provided, including links to useful sites, books, general camera set up, gear, editing software and relevant social networks.
Participants to bring: notebook, camera and lens/es.
The venue is fully accessible. However, there will be two sessions filming out in the town. Extra gear, etc can be left in the locked workshop room.


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It was the first to be awarded outdoors and the first outside of Stockholm.
It was the first economics prize awarded to a student (Paul) with his disseration adviser (Bob).
It was the first awarded jointly to two people living across the street from one another.
Paul became the first to give TWO Nobel prize lectures, having already lectured on behalf of Vickrey in 1996.
The backyard ceremony was held behind a residence that had housed two economics laureates (Paul Milgrom & Joe Stiglitz), plus 3-time SuperBowl winning coach Bill Walsh!
Also ununsual is that, by focusing on Paul's auction contributions, the Nobel Committee had left unmentioned ten of Paul's twelve most highly cited publications.
Paul's and Bob's Nobel lectures are available on YouTube here for Paul and here for Bob.


Our art practice encompasses a variety of techniques and media from photography to photo-based digital painting to more recently, encaustics and mixed media. Our passion for creating fine art overlaps nicely with our passion for travel, hiking, biking, kayaking and exploring the world, its unique people and natural and man-made beauty. We invite you to join us on this journey of adventure, exploration and creativity.
Note: All images are copyrighted and All Rights are Reserved. We do not allow usage without written authorization from us. If you would like to license one of our photos, please contact us at ray@rk2studio.com.
Images from the galleries above are organized by collection. Click on a collection below to view by subject matter.


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Q: Fix access path to Team Foundation in Visual Studio I created a new project and a repository on VSTS. When I tried to check it in, I'm getting the following error message.
Looks like it's trying to access the wrong folder under the wrong profile. "Owner" was a temporary account I had created while I was setting up my computer. Since then I deleted it.
It should be looking under my profile. How do I fix this? Whatever I click on this dialog is giving me an error. Is there a file that I can edit to fix this?

This is the error I get when I click Advanced:

When I try to manually configure my workspace, this is the error I got:

Any idea how to fix this? Thanks.

A: Similar problem was here and here. Maybe you have to check permissions of current user to folders on your errors or create server workspace:

redpajama_set_name: RedPajamaStackExchange


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BAA Enters New Partnerships with Broadway Grand Rapids & MSU's Wharton Center for Performing Arts
December 16th, 2019 | By Ryan Gilbert
If you seek Broadway on this pleasant peninsula, look about you! Broadway Across America (BAA) has entered into new partnerships with Broadway Grand Rapids and Michigan State University's Wharton Center for Performing Arts. BAA, part of The John Gore Organization, will work with each presenting organization to provide support with programming, operations, ticketing and marketing for touring Broadway engagements.
"Our goal is to bring Broadway to fans and fans to Broadway" says John Gore, Owner and CEO of BAA's parent organization, The John Gore Organization. "We are thrilled to be working with our new partners in Grand Rapids and East Lansing to bring the very best of Broadway to Michigan audiences."
"Our partnerships throughout North America deliver the very best in live entertainment to theater fans in their hometowns and provide a positive impact to the communities we serve," said Lauren Reid, Chief Operating Officer of The John Gore Organization. "Both Wharton Center for Performing Arts and Broadway Grand Rapids have vibrant and growing arts communities, and we couldn't be more excited to begin work together."
The Fifth Third Bank Broadway Grand Rapids' 2019-2020 season remains unchanged and will continue with Hamilton (January 21 – February 9, 2020), Roald Dahl's Charlie and The Chocolate Factory (March 10 – 15, 2020) and Escape to Margaritaville (May 26 – 31, 2020).
"Partnering with the preeminent producer and presenter of touring Broadway, BAA, offers us an unrivaled level of support for the work we do, allowing us to provide a range of exciting new services and benefits to West Michigan audiences," said Meg Goebel, Chair of the Board for Broadway Grand Rapids.
Additionally, Broadway at Wharton Center's 2019-2020 season remains unchanged and continues with My Fair Lady (February 26 – March 1, 2020), Wicked (April 1 – 19, 2020) and Dear Evan Hansen (June 16 – 21, 2020).
"I am very excited for MSU to partner with my friends and colleagues at BAA, having known many of them for decades." said Mike Brand, Executive Director of Wharton Center for Performing Arts. "MSU will benefit from having access to the state of the art software and strategies that BAA has developed to help make smarter decisions when programming and promoting Broadway shows. When considering bringing a partner in to work with us at MSU, BAA was the perfect fit."
Broadway Across America is part of The John Gore Organization family of companies, which includes Broadway.com, The Broadway Channel, BroadwayBox.com and Group Sales Box Office. Led by 14-time Tony-winning theater producer John Gore (Owner & CEO), BAA is the foremost presenter of first-class touring productions in North America, operating in 47 markets with over 4750,000 subscribers. Presentations include Disney's The Lion King, Wicked, The Book of Mormon, The Phantom of the Opera and Hamilton. Current and past productions include Ain't Too Proud, Beautiful, Cats, Chicago, Dear Evan Hansen, Mean Girls, Moulin Rouge! and To Kill A Mockingbird.
The John Gore Organization is the leading developer, producer, distributor and marketer of Broadway theater worldwide. Under the leadership of 14-time Tony-winning theater producer and owner John Gore, its family of companies includes Broadway Across America, Broadway.com, The Broadway Channel, BroadwayBox.com, and Group Sales Box Office. The company presents shows in 47 cities across North America as well as on Broadway, Off-Broadway, London's West End, Japan, and China. It has won Tony Awards in every producing category as well as numerous other Drama League, Drama Desk and Olivier Awards.


Mint condition 2016 R1200RS with ALL the goodies! 5000 adult driven miles. Miles may increase as I still might ride it.
Bike has Premium package, BMW Akrapovic exhaust (stock exhaust included) BMW valve cover guards, BMW hard bags, BMW GPS, Denali lights, VStream windshield(stock shield included) and Givi tail bag.
Bike has been babied since day one. Never dropped or down, always garaged on battery tender. Break in service done by BMW dealer and oil changed at 3700 miles by dealer.


CGCA's Support Teacher of the Year has won the District Title!
CGCA Principal Mr. Steve Derr, Ms. Durdley, and SCCPSS School Superintendent Mr. Thomas Lockamy at the ISPY Celebration Banquet.
for the 2013- 2104 school year!
1 - Learn the safety rules by looking at the brochure, the handouts and the PowerPoint!
2- Watch the funny video below "All About the Bus," just for fun.
4- Present the PowerPoint for your grade level to your class and use the SCRIPT to narrate as you show it.
5- Have the students examine the school bus danger zones and discuss why these areas are so dangerous.
6- Show the videos - they are the same for Middle and High and there are none for Elementary. The videos might be OK for upper Elementary - you preview and be the judge.
7- Have your students create their own PowerPoints or Posters illustrating the bus safety rules they have learned. Have them include at least 5 rules in their product.
8- Have your students sign the verification form.
The names of all the Progress Students are read on the P.A. System every Wednesday during Morning Announcements! Along with the names something is said that refers to the hard work that Progress students do to maintain their elevated level of behavior. For example: "Progress Students do not complain about their work and try their best!"
Use 3 Tablespoons of the Angel-Devil mixture, add 2 tablespoons water in a mug and mix. Microwave it for 1 minute. Eat cake.
MENTOS & DIET COKE EXPLOSION LAB!
Get your creative staff members to do a favorite art project or craft with the students!
Students sign up and purchase these activities - space is limited so you must have the funds available when these activities are offered!
Select a Demonstration test - and click on "Login", no credentials are required.
Georgia Milestones is the new assessment for Georgia that takes the place of the CRCT, EOCT, 3rd, 5th and 8th Grade Writing tests.
The Learning Odyssey requires a student log-in. All CGCA students in grade 2- 8 have log-ins. The student log-ins can be found on the CGCA Shares drive. All passwords are password and the "school" is Savannah.
All teachers should of these grades should have their own account so that they can add content to their student's portfolio, assign tasks and generate reports of their students' performance.


Certain features, services and applications are network dependent and may not be available in all areas. Additional terms, condition and / or changes may apply. All features, functionality and other product specifications are subjected to change without notice or obligation. Contact your service provided for more details.
Your product is a sophisticated electronic device. We encourage you to familiarise yourself with the user guide and instructions provided with and online. Please note the product contains high precision sensors and other parts which could be scratched or otherwise damaged if not handled carefully.
All warranty information, product features are subject to change without notice.


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You are at:Home»News»Dynamic Discs EMAC Truth to bring old flight to new disc
Dynamic Discs EMAC Truth to bring old flight to new disc
By Zach Parcell on	 2.29.16 News
Signature discs are nothing new to the sport of disc golf. Heck, we've even seen plastic blends named after top professionals. For Dynamic Discs professional Eric McCabe, he gets the honor of having a disc named after him. This isn't another signature disc, it's the new Dynamic Discs EMAC Truth.
Ever since the release of the Dynamic Discs Truth, the popular midrange from the Emporia, Kansas based business has been topped with the signature of the 2010 PDGA World Champion. Over the last year or so McCabe, and others, have noticed the flight of the Truth has slightly changed. What began with flight ratings of 5, 5, 0, 2, has started to show a little turn and a touch less fade.
During a Periscope broadcast last week, McCabe stated that while he heard the concern from others about the change of flight, he was still throwing a stockpile of the early run Truths that flew as designed so it was hard to understand the true difference in flight. As McCabe dipped into a newer stock of Truths he did notice the flight has changed and thus began an effort to bring the Truth back to its roots.
After some design work and testing, the end result is the new Dynamic Discs EMAC Truth. The midrange has been sent to the PDGA for approval and will be available in stores soon. To learn more about the EMAC Truth we talked with the man who has his name on the disc, Eric McCabe.
News came out Wednesday afternoon that a new EMAC Truth was sent off to the PDGA for approval. What can you tell us about this new Truth?
"The 'New' Truth will be called, the EMAC Truth. The current run of Truth's have seemed to become a bit too understable for what I had initially wanted the midrange to fly like. The EMAC Truth will fly a bit more stable, not quite as OS as the Verdict, but somewhere in between the current Truth and Verdict. More like the original runs, or the Pre made in Sweden tooling ones."
The current Truth isn't going away, correct? Is this something that you'll continue to have in your bag along side the new EMAC Truth?
"That is correct. We felt the current Truth is still a great disc, it's just not what I wanted it to fly like. We've decided to change the numbers on the current Truth to 5, 5, -1, 1, which is closer to the actual flight of the disc. The EMAC Truth will have the original Truth numbers of, 5, 5, 0, 2.
I do plan to continue to bag the current Truth, for big turnover shots that will hold the right line and finish flat."
During a Periscope broadcast you mentioned that this in a sense is returning the Truth to what it was always intended to be and to better fit what you wanted it to be, correct? When did these discussions first begin?
"I first noticed the disc starting to turn right more and more about a year ago. At first, I didn't think too much about it, seeing how I had a stock pile of the pre made in Sweden ones. When I first noticed, I approached Rusco and Tomas and we began working out the details. It has been a long time coming, but in beyond excited about this new mold, and I feel everyone else will agree as well."
When you first got the EMAC Truths in for you to test and took them out to the course, what were your initial reactions?
"WOW! I couldn't believe how great it flew, and the fact that it felt about the same as far as the mold goes made me even happier! Tomas. Is. A. Genius."
Do these EMAC Truths feel the same as the current Truth or will people be able to tell a difference?
"Yes, they do feel very similar. The stock stamps will be very easy to tell the difference. My signature silhouette will be removed from the original Truth, and a new design for the EMAC Truth has been made. Another way you'll be able to tell the difference between the two molds is the engraving on the bottom. The original Truth will stay the same, and the EMAC Truth will include my signature engraved in the mold. The initial release will be in my personal favorite plastic, Lucid. We will also be releasing the EMAC Truth is all of our more popular plastics across the board, i.e. Fuzion, Fluid, Moonshine, etc…"
Let's talk releases. When will we see the EMAC Truths be available? The current Truth will still be available, but with a new stamp, correct?
"That is correct. The current run will have a new stamp, basically just removing my name and changing the flight numbers. I personally believe the current run of Truths will become collector items. As far as release of the EMAC Truth goes…I'll just say before the Glass Blown Open."
You've had signature discs many times in your career. Now you have a disc NAMED after you. Is that a special feeling?
"Yes. It is actually an unbelievable feeling, I'm honored, grateful and simply overwhelmed at the support Jeremy Rusco and Dynamic Discs has shown me over the years. I never thought I would actually have a disc named after me, so to say it's a special feeling is an understatement."
Thanks to McCabe for the conversation. We were certainly surprised to learn of this news last week and had to learn more about how it all came together.
We've previously noticed the change in flight of the Truth and can't wait to see how the new EMAC Truth compares to those original Truths from a few years ago. Stay tuned to ATDG for more!
Fundraising for 2016 St. Jude Disc Golf Charity Invitational continues
David Wiggins Jr. blows away distance world record, throws over 1,100 feet


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Tag: construction vest
What's the deal with construction vest?
It's a long-standing practice for construction vest companies to erect giant concrete slabs, which, for the most part, sit in places that are too dangerous for humans to traverse.
In New York, that includes the Bronx, where a new vest company, Geringer Construction, is making it easier for construction workers to walk around the streets of Manhattan.
The firm, which will open a new store on the Lower East Side this spring, is also launching a website to help people get started in the field.
Geringers vest is a vest made of concrete.
The concrete slab it sits on is made of steel mesh that's held together by an electrical conduit.
The mesh connects to a conveyor belt, which drives a giant mechanical arm that pulls a large concrete slab into place.
As you walk through the vest, the arm makes a mechanical sound that you hear, which sounds like a clanking mechanism.
You can see the vest in action on Geringering's website.
A vest is generally made of a slab of concrete, a few pieces of plywood, and some wires and pulleys.
But Gering is making one that is made from a combination of steel, steel mesh, and electrical conduit—a combination that allows for a lot of flexibility.
Gers vest can hold up to 400 people and is built for people who are taller than 6 feet tall.
That's a big improvement over previous vest designs, which often required people to be standing on a very steep incline, with little room to walk and a lot to move around.
Gingerbread is building a more lightweight version of its construction vest that can hold as many as 100 people, but its vest is much smaller than the Gering product.
The Gering vest is about the size of a normal pair of jeans, and it has a built-in speaker that can be used to play music.
Gerbread has built a large vest that's about the same size as a normal vest, but it's smaller than what the company typically sells.
Gereing has built an all-in-one construction vest called the Gerenger vest, which includes a microphone, speaker, and a belt that attaches to the vest.
Genders vest is made by Gering.
Here you can see a vest that is similar in size and features to a Geringere vest.
The vest has a microphone that can also be used for music.
It's like a large-sized DJ booth.
The speakers can be mounted on a shelf and a pair of speakers can sit in the center of the vest for music playback.
When people walk through a Gerender vest, they hear a mechanical clanging sound that can sound like a motor running.
And it can make that clanging noise for about 10 to 15 seconds before the sound disappears.
The video below shows a vest GeringER vest that has a sound system attached to it.
Gergner has built its own audio system that it uses to send music from the speakers to the Gers earpieces, which are placed near the vest and in front of the wearer's ear.
You have to walk through it to get to the speakers, but once you get there, the Gerbere vest sounds pretty good.
Gebers vest, as shown, has speakers attached to the front and the rear.
Here, you can clearly see the speakers.
You could also use your voice to control the Geberer vest, using the Gercher voice.
You don't need a Gerchers vest, because the Gergers vest works with an app called VoiceOver.
VoiceOver uses a microphone and a speaker that sit next to the wearer and to the left and right of the speaker.
It works as an audio recorder.
It also can be controlled via a smartphone.
Gerchereve is a project that Gering and Gerbinge are working on together.
The two companies are using their own proprietary voice recognition technology to help build their products, Gebes VoiceOver being a way to communicate between the two companies.
Gegers voiceover has a lot in common with the Gertler system, which was developed by Gerns group in Germany.
But its strengths are that Gertlers voiceover can be made faster than other voice recognition systems, and the system has built-ins for speech recognition, voice search, and gesture recognition.
This system is also used by Apple in the iPhone and iPad, and is one of the reasons Gebings voiceover is able to connect to Apple's Siri system.
In addition to the voice recognition and gesture support, Geregers voice over can also send text messages and play music from a phone or tablet.
In order to make voiceover work, Gerbewear had to develop its own technology to create the sound that the Gerer vest creates.
Gern's voiceover system is designed to work with a device that's attached to
Posted on August 5, 2021 Categories DesignTags boldt construction, construction vest, gingerbread construction company, messer construction
How to construct your own construction vest, contract template and construction contract
A construction vest is a simple, low-cost, and easy to follow way to legally construct a building in your own name.
It is also a great way to build a small business.
In this article, we'll show you how to construct a construction vest in your name, using templates for a building contract, a construction contract, and a construction template.
Read on for instructions.
How to Construct a Construction Vest in Your Name Construction vest templates The construction vest template can be found on a construction site's website or through a search engine.
It may be helpful to take a look at the template to understand the layout of the contract and construction vest requirements.
In addition, you may want to read the following articles to learn more about the construction vest.
Construction Contract Template The construction contract template is a PDF file that can be downloaded and printed.
It can be printed on most printer-friendly paper and it's compatible with most printing software.
It's useful to have the template for the construction contract ready when you create the construction contracts and construction contracts templates.
This template is available in a range of sizes and the construction costs of the project can vary depending on the size of the contracts and templates you use.
You can download the construction template here: Construction Contract PDF Construction Contract Templates There are a number of different templates that can work for the projects we've described so far.
In most cases, you'll need a construction contractor to perform the work.
You'll want to hire a construction company to construct the project.
To build a construction project, you can hire contractors, and you can pay for them with your own money.
The construction contractors are usually based in a different state and often provide you with a different type of work.
For example, a contractor working for the government may provide services for you in your home state.
You may need to pay them a commission for doing the work they provide.
You might also need to buy a property in your state and build it.
This is how the construction projects are usually built: In the construction project template, you need to choose a location, set a starting time, and an amount of money to cover the construction cost.
The contract will need to include a specific date, time, cost, and location.
The starting time is also important, as the contractor will need a set amount of time to complete the project and set the start date.
You will also need a building envelope for the project to secure the work to.
You need to give the contractor a specific amount of the construction materials to complete.
The contractor will then work on the project from the beginning to the end.
Once completed, the contract will be signed by the builder and the building contractor will leave the project with a completed contract.
A construction project is usually built in the same building that you'll build the home.
The template will tell you which state you're working in, the building envelope, and where you can find your building envelope.
You also need specific information about the materials you're buying to get started.
You're not looking to buy cheap materials and you're not asking the contractor to provide you a lot of them.
You should be able to pay for your own materials yourself, but you should pay a minimum price per square foot (sqft) that covers your cost of materials.
If you're building a small home, you might want to ask the contractor if you can buy your materials for less than the standard building envelope price per sqft.
If so, you should ask your contractor for a price range to negotiate for your materials.
The building envelope can be the contract's cover sheet.
It tells you what materials are going to be used for the contract.
If the contract includes a clause that says "the contractor must cover the costs of this work", then you'll want the contractor's name on the cover sheet to provide the contractee with some legal protection.
You don't need to list your name in the contract; your name may be listed elsewhere on the contract as part of the contractor.
If your contractor is a construction subcontractor, you're going to need to write a separate contract covering that work.
If a contractor's work is the sole responsibility of the builder, then the builder can ask for an additional payment for their work.
In our example, we've already paid for a lot more of the work than the construction envelope.
We paid for everything except the roof and the roof deck, but the contractor still needs to pay.
The amount of work to be done for a single square foot of land varies depending on your construction contract.
For a larger building, like a condo building, you'd want the builder to pay a larger price per unit of work than for a smaller one.
If that's the case, you could ask the builder if you want to be responsible for paying the contractor for the cost of your materials and building work.
The builder's name will be included on the covering sheet for the
Posted on July 17, 2021 Categories ConstructionTags construct, construction contract template, construction memes, construction vest


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Home News Childrens Health News RSNA: Overweight Tied to Worse Brain Health in Preteens
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RSNA: Overweight Tied to Worse Brain Health in Preteens
Findings include worse brain white matter microstructural integrity, reduced cortical grey matter thickness, and decreased functional connectivity
By Lori Solomon HealthDay Reporter
THURSDAY, Dec. 8, 2022 (HealthDay News) — Higher weight in childhood is associated with worse neurodevelopmental outcomes, according to a study presented at the annual meeting of the Radiological Society of North America, held from Nov. 27 to Dec. 1 in Chicago.
Simone Kaltenhauser, Ph.D., from Yale School of Medicine in New Haven, Connecticut, and colleagues assessed the relationship of preadolescents' weight and body mass index (BMI) with microstructural, morphological, and functional brain magnetic resonance imaging metrics. The analysis included data from 5,169 children (aged 9 to 10 years) from 21 U.S. centers (overweight, 21 percent; obese, 17.6 percent).
The researchers found that higher weight and BMI-z-scores were associated with lower fractional anisotropy (FA) values in 16 and 25 tracts, higher FA values in one and no tracts, lower axial diffusivity values in 23 and 30 tracts, higher radial diffusivity (RD) values in five and 10 tracts, lower RD values in three and no tracts, lower neurite density (ND) values in 11 and 13 tracts, and higher ND values in five and one tracts, respectively. FA reductions were most pronounced in the corpus callosum, fornix, and (parietal and temporal) superior longitudinal fasciculus for both weight and BMI-z-scores. With increased weight and BMI-z-scores, thinning of 63 and 54 cortical regions, higher surface area of 52 and 26 cortical regions, and significant disruptions in 37 and 31 internetwork and intranetwork correlations, respectively, were seen.
"Increased BMI and weight are not only associated with physical health consequences but also with brain health," Kaltenhauser said in a statement. "Our study showed that higher weight and BMI z-scores in 9- and 10-year-olds were associated with changes in macrostructures, microstructures, and functional connectivity that worsened brain health."


This is such awesome advice, as I'm sure I'll have to be answering these questions in the next couple years!
What a wonderful, insightful post. Our oldest (who is autistic) does not know that he is. He knows that he is different but doesn't know why, and the rare times he asks, I let him know that he sees the world differently than we do, and give positive examples to show him. I am afraid telling now that he is autistic would be detrimental, so we'll hold off on the full explanation until he is older. Maybe we need to start sugar-coating it for the family members who are still unaccepting/in denial about the diagnosis, because straight up telling them hasn't done anything. Teachers, at least, understand and care. That helps.
I haven't read this book, but statements like, "[S]he examines so-called brain "problems" including learning disabilities, autism, ADD, depression, anxiety and bipolar disorder and demonstrates how they can be directly associated with the potential for great talent," worry me. It comes very close to the idea that people with disabilities are only valuable if they have other hidden talents. Not everyone with autism is a secret genius. Some kids have intellectual disabilities and physical disabilities that will limit them in nearly every way. For some people bipolar disorder or depression will lead to early suicide. I think it can be dangerous to focus on "look at all the special stuff that disabled people can do" because it can lead to devaluing people who can't do those things. Everyone has inherent value as a human, even if they can't do math, or puzzles, or run, or sing or paint.


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Microsoft Unveils Limited Edition Greaseproof PUBG Xbox One Controller
Ubergizmo Tyler Lee Sep 6, 2018
Ubergizmo Sep 6, 2018
Tyler Lee Sep 6, 2018 Read Source
While Tencent's PlayerUnknown's Battlegrounds (PUBG) has been around for a while now, it appears that as far as the Xbox One is concerned, the game has only recently managed to achieve a full release. To celebrate that fact, Xbox Australia announced a very limited edition greaseproof Xbox One controller, presumably for all your chicken dinners.
According to Xbox Australia, the controller will feature a coating of urethane that was applied by hand. This not only makes the controller greaseproof, but also resistant to scratches and water. It will also feature a unique grey, black, and yellow colorway, and so far it seems that there are only plans to release 10 of these, and that they will be limited to those in Australia.
It will also only be available by entering a competition, in which the rules, terms and conditions, can be found on the Xbox ANZ Facebook page. Interestingly enough this is not the first time that we're seeing Xbox Australia come up with weird and cool marketing campaigns. Last year the company launched the Xbox Inflatable for the pools, and a couple of years ago there was also the Xbox Onesie.
That being said for those who won't be able to take part in this, not to worry because Microsoft had previously announced another PUBG controller. This will be limited too, although we reckon it will be less limited than 10 units.
Microsoft Unveils Limited Edition Greaseproof PUBG Xbox One Controller , original content from Ubergizmo. Read our Copyrights and terms of use.