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Disclosed are compounds consisting of a plurality of nucleosides which are covalently linked by at least one aminoalkylphosphoramidate linkage of the formula ##STR1## wherein n=2 to 6 and Nu1 and Nu2 represent nucleoside phosphates.
1. A compound consisting of a plurality of nucleosides selected from the group consisting of adenosine, thymidine, cytosine, and guanosine, wherein the nucleosides are covalently linked by mono-phosphodiester bonds and one or two aminoalkylphosphoramidate linkages of the formula: ##STR5## wherein n is 6, and Nu1 and Nu2 represent nucleoside phosphates wherein the nucleosides are as stated above, and
wherein Nu1 +Nu2 is 17 nucleotides.
Work described herein was supported by cooperative grant U01 124846 from the National Institute of Allergies and Infectious Disease and a grant from the G. Harold and Leila Y. Mathers Foundation.
There is at present growing interest in non-radioactively labelled modified oligodeoxynucleotides. Biotin (Agrawal. S. et al., Nucleic Acid Research, 14:6227-6245 (1986); Agrawal, S. Tet. Lett., 30:7025-7028 (1989)), florophores (Cardullo, R. A. et al., Proc. Natl. Acad. Sci. USA, 85:8790-8794 (1988); Agrawal, S. et al., J. Cell Biology, 107:468 (1988); Haralambidis, J. et al., Nucleic Acids Res., 18(3):501-505 (1989)), intercalating (Helene, C. and J. J. Toulme, Oligodeoxynucleotides-Antisense Inhibitors of Gene Expression, Ed. J. S. Cohen, Macmillan Press, 137-166 (1989)) and chelating (Oser, A. et al., G. Nucleic Acid Research, 16:1181-1196 (1988)) reagents attached to synthetic oligonucleotides are becoming important tools of molecular biology. A variety of enzymatic and chemical procedures have been developed for their synthesis (Matthews, J. S. and L. J. Kricka, Anal. Biochem., 169:1-25 (1988)). Central to some of these procedures are (a) the introduction of a reactive group at either the 3'- or 5'- terminus of the oligonucleotide (Agrawal. S. et al., Nucleic Acid Research, 14:6227-6245 (1986); Agrawal, S. Tet. Lett., 30:7025-7028 (1989); Fidanza, J. A. and L. W. McLaughlin, J. Am. Chem. Soc., 111:9117-9119 (1989); Nelson, P. S. et al., Nucleic Acid Research, 17:7187-7194 (1989)) or (b) the synthesis of modified nucleosides which contain the masked reactive group and are incorporated into the nucleic acid (Fidanza, J. A. and L. W. McLaughlin, J. Am. Chem. Soc. 111:9117-9119 (1989)). The presently-available methods are useful, but are limited in their usefulness for site specific internal non-radioactive labelling of synthetic oligonucleotides possible.
The present invention relates to a method of site specific functionalization of oligodeoxynucleotides for non-radioactive labelling, as well as to functionalized oligodeoxynucleotides and non-radioactively labelled oligodeoxynucleotides produced by the method. This method makes it possible to modify one or more selected internucleoside phosphate(s) in a synthetic oligodeoxynucleotide in such a manner that it (they) can be used to incorporate a non-radioactive material into the synthetic oligodeoxynucleotide. In particular, the method of the present invention is used to modify one or more selected internucleoside phosphates in a synthetic oligodeoxynucleotide , to give aminoalkylphosphoramidate residues or aminoalkylphosphotriester residues. The amino group(s) of the resulting modified residue(s) is then further reacted with a non-radioactive label, such as biotin, fluorescein or rhodamine (e.g., N-hydroxysuccinimide ester of biotin, N-caproyl amidobiotin, and a variety of fluorophore isothiocyanates), to produce a non-radioactively labelled oligodeoxynucleotide in which the label is present at a predetermined location or locations.
In the present method, an H-phosphonate internucleoside linkage is oxidized with an appropriately protected diamine, such as N-1-trifluoroacetylhexanediamine, (CF3 CO NH(CH2)6 NH2), in the presence of an appropriate solvent, such as carbon tetrachloride, to give a phosphoramidate internucleoside linkage (Zwierzak, A., Synthesis, 507-508 (1975); Froehler, B. et al., Nucleic Acid Research, 16:4831-4839 (1989); Letsinger, R. L. et al., J. Am Chem. Soc., 110:4470-4471 (1988); Agrawal, S. et al., Proc. Natl. Acad. Sci. USA, 85:7070-7083 (1988); Jager, A. et al., Biochemistry, 27:7237-7246 (1988)). The resulting phosphoramidate internucleoside linkage is stable under oligonucleotide assembly conditions using phosphoramidite chemistry (Caruthers, M. H. et al., Methods in Enzymology, 154:287-313 (1987)) and to subsequent deprotection steps. Alternatively, the H-phosphonate internucleoside can be oxidized, to give a phosphotriester internucleoside linkage, with an appropriately protected aminoalkyl alcohol in N-methylimidazole-triethylamine-carbon tetrachloride (e.g., 5:5:90).
The present method can be used to produce non-radioactively labelled oligodeoxynucleotides which include a non-radioactive material at one or more sites and are useful in research and in the diagnosis and treatment of diseases and conditions of interest.
FIG. 1A is a graphic representation of the results of analytical ion exchange HPLC of oligomers 1 (chromatograph (a)), 2 (chromatograph (b)), and 5 (chromatograph (c));
FIG. 1B is a graphic representation of the results of reversed phase HPLC of ion exchange purified oligomers 1 (chromatograph (d)), 2 (chromatograph (e)), and 3 (chromatograph (f));
FIG. 2A is a graphic representation of the results of reversed phase HPLC traces of oligomer 2 (chromatogram (a)), and reaction mixture of oligomer with biotin N-hydroxysuccinimide (chromatograph (b)); and
FIG. 2B is a graphic representation of the results of reversed phase HPLC traces of oligomer 5 (chromatograph (c)) and reaction mixture of oligomer 5 with biotin N-hydroxysuccinimide (chromatograph (d)).
The present invention relates to a method of producing oligodeoxynucleotides which have a desired (selected) nucleotide sequence and which are labelled internally with a non-radioactive material or reporter group at one or more internucleoside linkages. In the method of the present invention, one or more selected internucleoside phosphate residues are modified to produce aminoalkylphosphoramidate residues or aminoalkylphosphotriester residues which are present in an oligodeoxynucleotide at selected positions. The amino group(s) in such modified (functionalized) residues is further reacted with a label or reporter group, resulting in production of a non-radioactively labelled oligodeoxynucleotide labelled internally at selected location(s).
Briefly, the present method is carried out by oxidizing an H-phosphonate internucleoside linkage using an appropriately protected diamine, represented by the formula XCONH(CH2)n NH2, in which X is a base labile protecting group and n can be 2 or more. For example, an H-phosphate internucleoside linkage is oxidized using N-1-trifluoroacetylhexanediamine (CF3 CONH(CH2)6 NH2) in the presence of an appropriate solvent, such as anhydrous carbon tetrachloride. As a result, a primary aliphatic amine is incorporated at the internucleoside phosphate as phosphoramidate. In the case of phosphotriester linkages, oxidation is carried out using a suitably protected amino alcohol, represented by the formula XNH(CH2)n OH, in which X is a base labile protecting group and n can be 2 or more. For example, an H-phosphonate internucleoside linkage is oxidized using N-1-fluoroenylmethyoxcarbonylaminohexanol F-MOC-NH(CH2)6 OH in the presence of N-methylimidazole-triethylamine-carbon tetrachloride. The remaining nucleotides needed to produce the desired nucleotide sequence are added using art-recognized techniques, such as phosphoramidate, H-phosphonate chemistry or methyl phosphoramidate. (Caruthers, M. H. et al., Methods in Enzymology, 154:287-313 (1987); co-pending U.S. patent application Ser. No. 07/334,679 (Method of Synthesizing Oligonucleotides and Their Analogs Adaptable to Large Scale Synthesis, by S. Agrawal and P. Zamecnik, filed Apr. 6, 1989, the teachings of which are incorporated herein by reference; Agrawal, S. and J. Goodchild Tet. Lett., 28(31):3539-3542 (1987)). After the desired oligodeoxynucleotide is produced, the protecting group present on the primary aliphatic amine is removed. The unmasked amino group can now react with one or more selected labels or reporter groups. As a result, the oligodeoxynucleotide is labelled, non-radioactively, at one or more selected internal locations. One or both amino groups present in the diamine react with the selected label.
The method of the present invention is represented in a series of steps below. The following is an explanation of those steps, with reference to the respective reactants and steps represented below. ##STR2##
1. Initial coupling of two nucleotides (designated Nu1 and Nu2) is carried out, using H-phosphonate chemistry. Generally, Nu1 is bound to a solid support, such as CPG and terminates in a diemthoxytrityl residue (designated X). As a result, a support-bound dinucleoside H-phsophonate is produced (designated (II)).
2. The support-bound dinucleoside H-phosphonate (II) is subsequently oxidized by being combined with an appropriately protected diamine in the presence of a suitable solvent, resulting in formation of a phosphoramidate internucleoside linkage or a phosphotriester internucleoside linkage (and linking of the protected diamine to the dinucleoside through the unprotected amino group of the diamine). The resulting product is designated (III).
3. The dimethoxytrityl residue present on the unbound end of (I) is removed and the remaining deoxynucleotides of the desired oligodeoxynucleotide to be produced are added at the now free end, using phosphoramidate chemistry or H-phosphonate chemistry, producing a support-bound oligodeoxynucleotide (IV) which includes the phosphoramidate linkage produced in step (2).
4. The protecting group Y present on the diamine is removed and the compound is removed from the solid support. This results in production of an unbound functionalized oligodeoxynucleotide (i.e., an aminoaliphatic oligomer or an oligodeoxynucleotide having a desired nucleotide sequence and an alkyl amino group present at the selected internucleoside phosphate(s) as a phosphoramidate or phosphotriester).
5. The unbound functionalized oligodeoxynucleotide is reacted with an appropriate form of a non-radioactive material, which becomes bound to the amino group and serves as a label or reporter group on the oligodeoxynucleotide. This results in production of an oligodeoxynucleotide labelled site specifically with a non-radioactive material. The non-radioactive material can be a fluorophore, a spin label, an enzyme, a chelator, a heterocyclic molecule, a protein, a lipid, a drug derivative, an antigen, an intercalator or other organic or inorganic moiety.
In a specific embodiment of the present invention, which has been used to produce non-radioactively labelled oligodeoxynucleotides, the following steps were carried out to produce a non-radioactively labelled oligodeoxynucleotide:
1. Initially, (I) and Nu2 were coupled, using art-recognized H-phosphonate chemistry, resulting in production of a support-bound dinucleoside H-phosphonate.
2. The support-bound dinucleoside H-phosphonate was oxidized, using N-1-trifluoroacetyldiaminohexane (NH(CH2)6 NH-CO-CF3) in carbon tetrachloride-dioxane, resulting in formation of a phosphoramidate internucleoside linkage between Nu2 and Nu1.
3. The remainder of the nucleotide sequence of the oligodeoxynucleotide was produced in a two-step procedure in which the dimethoxytrityl residue [DMTrO] was removed from the nucleotide now bound to the solid support (Nu2 in the reaction scheme above) and the desired nucleotides were added stepwise (i.e., to the now free end of the dinucleoside which, for convenience, can be referred to as the 5' end).
4. The protecting group (-CO-CF3) present on NH(CH2)6 NH-CO-CF3 was removed during deprotection of oligonucleotides in aqueous ammonia, resulting in production of a functionalized oligodeoxynucleotide (one in which the previously protected amino group is unprotected) of the desired sequence, in which there is an aminoalkylphosphoramidate residue of the formula ##STR3## present at the desired internucleoside phosphate linkage(s).
5. The unbound modified oligodeoxynucleotide with the aminoalkylphosphoramidate residue was reacted with a non-radioactive material, such as biotin, fluorescein or rhodamine in appropriate form (e.g., N-hydroxysuccinimide ester of biotin, N-caproyl amidobiotin, fluorophore isothiocyanates), which becomes bound to the amino group of the aminoalkylphosphoramidate internucleoside linkage.
Alternatively, a functionalized oligodeoxynucleotide of the desired sequence, in which there is an aminoalkylphosphotriester residue present at the desired internucleoside phosphate linkage(s) can be produced. In this case, the non-radioactive material becomes bound to the amino group of the aminoalkylphosphotriester internucleoside linkage.
The production of oligodeoxynucleotides labelled at a selected site or sites by the present method is described in greater detail in the Exemplification.
It is possible, using the present method, to produce oligodeoxynucleotides of desired sequence which are labelled internally at one or more nucleosides. The oligodeoxynucleotide backbone can be unmodified (e.g., as it occurs in nature) or modified (e.g., amidate, methylphosphate, phosphothioate, phosphotriester backbones). The label present at two or more sites can be the same (e.g., biotin) or different and can be present at as many sites as desired. As described in the Exemplification, a 17-met of the sequence shown has been produced, functionalized at a selected site or sites and labelled at the site(s) with a non-radioactive material.
In the above description of the present method, two single nucleotides (designated Nu1 and Nu2) are initially joined using H-phosphonate chemistry and the resulting oligodeoxynucleotide is functionalized at the internucleoside phosphate linkage formed between Nu1 and Nu2. However, any number of nucleotides can be joined, using art-recognized techniques such as H-phosphonate chemistry, before modification of a selected internucleoside phosphate linkage is carried out. This has been carried out, as described in the Exemplification, to produce a 17-mer functionalized at a central internal site (oligomer 3) and a 17-mer functionalized at two internal phosphate linkages (oligomer 5). Using H-phosphonate chemistry, for example, an internal nucleotide (e.g., a support-bound nucleotide such as Nu1) can be added to, resulting in production of a longer sequence (e.g. Nu10 Nu9. . . Nu1). The longer sequence can then be functionalized, by the method described above, resulting in production of a functionalized oligodeoxynucleotide (e.g., Nu10 ↓Nu9 . . . Nu1, in which the internucleoside phosphate linkage between Nu9 and Nu10 is modified). The modified oligodeoxynucleotide can then be further elongated by addition of selected nucleotides to produce a modified oligodeoxynucleotide of desired sequence. The protecting group present can be removed, as described above. Alternatively, the functionalized oligodeoxynucleotide initially produced can be extended (by addition of selected nucleotides), one or more additional internucleoside phosphate linkages can be modified and a functionalized oligodeoxynucleotide which has two or more sites at which non-radioactive material can be added is, thus, produced.
Protecting groups other than trifluoroacetyl (--CO--CF3), as described above, can also be used to protect the diamine. Other base labile protecting groups, such as F-moc and t-boc, may also be used. The linker present between the two amino groups in the diamine used can be of any suitable length (e.g., --(CH2)2 -- to --(CH2)n --); the length used in a particular case can be determined empirically. The diamine can be branched or unbranched and bi-functional or multifunctional.
N-1-trifluoroacetylhexanediamine, CF3 CONH(CH2)6 NH2, was prepared by adding ethyltrifluoroacetate (1.2 ml, 10 mmol) dropwise over one hour to a stirred mixture of hexanediamine (1.16g; 10 mmol) and triethylamine (1 ml; 7 mmol) in 20 ml methanol. The solution was stirred overnight. After removal of solvents, the reaction mixture was flash chromatographed on silica using 0-25% methanol in dichloromethane. The fractions containing the desired product were pooled and concentrated to give a colorless powder (1.1 gm, yield-42.6%) , M.pt. 52°. 1 NMR (CDCl3, d, TMS=0.00) 7.1-7.2 (m 3H, NH2, NH) 3.2-3.3 (m 2H CO-NH-CH2) 2.8-2.9 (m 2H CH2 -NH2) 1.2-1.6 (m 8H - CH2 - (CH2 )2 -CH2).
To test the efficacy of (CF3 CONH(CH2)6 NH2) for amino group introduction at specific sites of oligonucleotides, a 17-mer sequence, GTA AAA CGA CGG CCA GT, (oligomer 1) was made. Oligomers (designated 2-5 below) carrying aminohexyl residues at different sites, as shown by (↓), were also made.
______________________________________Oligomer # Sequence______________________________________1 GTA AAA CGA CGG CCA GT2 GTA AAA CGA CGG CCA G.sup.↓ T3 GTA AAA CG.sup.↓ A CGG CCA GT4 G.sup.↓ TA AAA CGA CGG CCA GT5 G.sup.↓ TA AAA CGA CGG CCA G.sup.↓ T______________________________________
The steps involved for labelling, for sequence 2, are shown below: ##STR4##
The first coupling (represented by (a) in the above reaction) was carried out using H-phosphonate chemistry. This resulted in a production of a support-bound dinucleoside H-phosphonate (II), which was then oxidized with 4% N-1-trifluoroacetyldiaminohexane (I) in carbon tetrachloride-dioxane (8:2, v/v) for 30 minutes, resulting in production of (III). After oxidation with CF3 CONH(CH2)6 NH2 (step (b) of the above reaction), the assembly of the rest of the oligodeoxynucleotide sequence was carried out (step (c)) using phosphoramidite or H-phosphonate chemistry. This resulted in production of the oligonucleotide (IV), which was deprotected in aqueous ammonia for 6 hours at 55° C. (step (d)), resulting in formation of the aminohexyl oligomer (V).
Assessment of the oligomers 1-5 was carried out. Analytical ion exchange HPLC of oligomer 2 showed the major peak eluting earlier than that of oligomer 1 with the same gradient (FIG. 1a and 1b), confirming that in oligomer2, one of the internucleoside linkages is a phosphoramidate linkage, which is non-ionic at phosphores. Sequences 3 and 4, which are functionalized at different sites, also showed a HPLC profile similar to that of oligomer 2. Oligomer 5, which is functionalized at two sites, was eluted even earlier (FIG. 1A, chromatograph (c)).
When ion exchange HPLC purified oligomer 2 was checked on reversed phase HPLC, it gave a doublet peak in ratio of 1:2 (FIG. 1A, chromatograph (e)) compared to 1 (FIG. 1A, chromatograph (d)). This results from the diastereoisomeric nature of phosphoramidate internucleoside linkage. Similarly, oligomer 5 eluted as a broad peak because of two such diastereoisomeric linkages (FIG. 1f). Both oligomers 2 and 5 had retention times longer than that of oligomer 1 because of the hydrophobic nature of the alkyl chain present in oligomers 2 and 5.
Reaction of oligomer 2 was carried out with biotin N-hydroxysuccinimide using reported conditions (Agrawal, S. et al., Nucleic Acid Research, 14:6227-6245 (1986)). The reaction mixture after gel filtration (Sephadex G-25) showed two new peaks of the diastereomeric biotin adducts (FIG. 2A, chromatograph (b)). Similarly, reaction of oligomer 5 gave a broad peak ass a doublet eluting later than the unreacted material (FIG. 2B, chromatographs (c) and (d)).
The method described herein provides a way for functionalizing oligonucleotides at one or more selected or specified sites. As described above, the subject method of introducing reporter groups has been carried out by reaction of the functionalized oligodeoxynucleotide with biotin active ester, resulting in production of an oligodeoxynucleotide labelled at the selected site(s) with biotin. In addition, the aminohexyl residue present was reacted in high yield with fluorescein and rhodamine isothiocyanate, to produce a fluorescent hybridization probe. Multiple labelling may increase the sensitivity of detection in diagnostic assays.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
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Whidbey Island Life
Puget Sound is home to dozens of islands. Sue Frause has lived on Whidbey Island since 1975 and still thinks it's fun taking a ferry to get home. Read about her island's colorful characters and what makes Whidbey such a unique place.
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Ragnar Relay Northwest Passage: Running 190 miles from Blaine to Langley
If you've never heard of the Ragnar Relay Series, it's returning to Whidbey Island. The Ragnar Relay is a 200(ish)-mile race where teams of a dozen race continuously day and night, relay style. While one runner runs, everybody else is on support duty in the race vehicles. The team has two vehicles, with runners 1-6 […] [Read More]
Postcard from Vancouver: Riots, not the Stanley Cup, are brought home to the city
I'm winding up my BC adventure, having spent a week in Vancouver, Whistler and now Victoria. Last night was a sad night for my favorite west coast city. Not only did they not bring home the Stanley Cup, Vancouver was trashed by a large group of hooligans. As the world watched, the city showed its […] [Read More]
Postcard from British Columbia: Following the Canucks for better or worse
Whistler, BC – A friend recently emailed me, concerned that I hadn't updated my Seattle P-I blog in a while: "Have not heard from you or anything about your trip. I keep checking your P-I blog to see what's going on, and no change. Are you OK? Is something wrong?" No, everything's fine and I'm […] [Read More]
Whidbey racks up awards in Evening Magazine's Best of the Northwest Escapes
I posted earlier that Whidbey Island was voted the #1 Best Island Excursion by Evening Magazine. Well, our rock has racked up an additional dozen awards. Here they are — congratulations to the winners! Whidbey Island #1 Best Local Island Excursion Admiralty Head Lighthouse #1 Best Lighthouse Deception Pass State Park #1 Best State Park […] [Read More]
Walk on the ferry and take the Sounder to Mariners and Sounders daytime games
I don't know too many islanders that thinks it's fun to drive into Seattle before an event, find a place to park (and pay), and then beat feet to make the boat back to the island. Sound Transit to the rescue! Sounder trains are again serving weekend Seattle Mariners and Seattle Sounders FC daytime games. […] [Read More]
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Working with color can be an amazing journey, and in Photoshop CS4 for Photographers: Creative Color, Brooks Institute of Photography professor Chris Orwig teaches a different way to create great color in photographs. Chris goes beyond the basics by showing how color can have a profound effect on a photograph's impact. He demonstrates adjusting color and tone to make images more vivid, changing colors, working with color saturation, cross processing, and more. The course culminates with real-world projects that utilize the techniques Chris has shown for creating intriguing and beautiful color photographs. Exercise files accompany the course.
Hey welcome! My name is Chris Orwig and I am going to be your host and guide on this training adventure and I am a photographer and I am a teacher. I am on the faculty of the Brooks Institute in Santa Barbara, California where we are right now and it is a beautiful winter morning this morning. There is something amazing to me about color. What we are going to do is talk about how we can work with colors in Photoshop from a photographic perspective. What I mean by that is we are going to talk about how we can work with color in order to create images of impact. Now what exactly are we going to cover in this training title? We are going to cover a wide range of topics, but just to illustrate, have you ever looked at a photograph and said, how do they create those just engaging, compelling, vivid, life-like colors? We are going to deconstruct how you can create vivid colors in your own photographs. We will also talk about how we can saturate our photos and how we can do some advanced saturation techniques. Now that's not all we are going to do.
We are going to go to the other side of the spectrum and take a look at how we can remove color and then perhaps tone our images or add a cross-processing effect. We will also take a look at how we could change colors or for that matter, create maybe an edgy color and tone effect. Now we are going to cover all of this and more and my hope is that throughout this training title that you will pick up a handful of tips and tricks, a lot of information, yet even more. That you will be inspired, that you will pick up a ton of inspiration for how you can work with color and how you can make your photographs come to life by understanding how color works and by understanding how you can work with color in order to create more compelling photographs.
All right, well, we have a ton of ground to cover, so without further delay, let's begin.
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Coming along well! I started from the back and I'm working forward. Don't ask.
I'm making this a bullpup, and I've modelled the buttstock after the OC-14 Groza (my favorite!) and I'm modelling the bullpup action after it as well:
I didn't get it in the pic, but I have a rear sight block partially converted for a pistol build I never completed. Well, a little modification and presto I have salvaged it for the "mounting block" that will support an AKSU-style hinged top cover. The cover was beat up some, and I had to peen and flex it back to some semblance of parallel sides.
As you can see, I'm nearly finished with the buttstock. The buttstock will have a rubber pad as soon as I find something suitable. Not sure where to get rubber that thick, and it is larger than all the rubber stock pads I have. The buttstock, when locked, keeps the top cover from coming loose in the back. Sling swivel is coming soon. The hinge didn't turn out exactly as I'd planned, but it works anyway so I'm not complaining... just looks a little odd!
YES - that is a home-built magazine well! It works! As you can see, I haven't wire brushed it or sanded it all nice and pretty yet. I haven't even pinned the latch retainer pin into place yet - kept it loose while I was fitting all the pieces in place!
It is flared to help magazine loading. It will need only a rear locking lug, the clearance is just enough so that I don't need anything on the front. The front spine of the well doubles to cover up the opening of the original magazine well... it was big enough that I didn't want it exposed.
That's a Saiga receiver cover plate, I just cut it down and riveted it in place to cover up the trigger opening.
I also spring loaded the firing pin:
I'm going to use the original Saiga sear and disconnector, and the bullpup will use an original trigger I had laying around. That puts the parts count up one more for me to 17 as I'm building it, but I'm covered any way you slice it with the buttstock, magazines, barrel, and muzzle brake. I think I have 9 US parts total.
Next on the list is chambering the barrel, adding the gas block, the bullpup assembly, and loading up some rounds


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The Nebraska Center for Cellular Signaling (NCCS) was formed in the fall of 2003 by Dr. Margaret Wheelock to create a Center of Biomedical Research Excellence (CoBRE) under the IDeA program, which is funded primarily by the National Institutes of Health (NIH) and the National Institute of General Medical Sciences. This Center, through the collaborations of the University of Nebraska Medical Center, Creighton School of Medicine, and the University of Nebraska-Lincoln, fosters new research initiatives dedicated to improving the oral and dental health of Nebraskans and citizens of other states. Junior and established researchers at these institutions conduct innovative and collaborative research efforts supported by the Center. This Center presents a strong mentoring environment to attract and promote the development of promising investigators.
A number of areas within the field of cell biology are rapidly converging on a common theme: cellular signal transduction. This is particularly true for the fields of cell adhesion, cell motility and cancer biology. The main focus of this Center is to bring together individuals studying signal transduction to form an organized, cohesive group that will provide leadership and mentoring to junior faculty interested in signaling.
Current Research Projects
The Mechanism of Aberrant DNA Methylation in Mouse Lymphomagenesis
Rene Opavsky, Ph.D., assistant professor, Eppley Institute, UNMC
The Functions and Regulatory Mechanisms of a Specific Protein Phosphatase 1 Complex in the DNA Damage Response and Cancer Progression
Aimin Peng, Ph.D., assistant professor, Oral Biology, UNMC
Live Cell Microscopy Core
The Live Cell microscope located at the College of Dentistry in Lincoln provides a humidity- and CO2 -controlled growth chamber and Intelligent Imaging Innovations software for collecting images. This system includes digital de-convolution, 3D rendering and lasers for FRAP analysis of protein turnover. This microscope is available for use by investigators at UNL or the COD. A second Live Cell microscope is housed in the Eppley Institute. This instrumentation is available to investigators in Omaha (UNMC and Creighton), and provides a humidity- and CO2 -controlled growth chamber and Intelligent Imaging Innovations software for collecting images, spinning disc confocal, digital de-convolution, 3D rendering and lasers for FRAP analysis of protein turnover. For more information or to schedule imaging, please contact the director: Jim Wahl, Ph.D. at 402-472-1324 or firstname.lastname@example.com.
External Advisory Board
Thomas Carey, Ph.D., University of Michigan
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Jean Schwarzbauer, Ph.D., Princeton University
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George Prendergast, Ph.D. Lankenau Institute for Medical Research (LIMR)
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Charity and Sponsorship Committee
We're committed to making a real difference to the communities we serve. One of the ways we do this is by supporting a wide range of charitable causes and community organisations.
Our Charity and Sponsorship Committee is made up of First people from across the North of England region. The committee considers and makes decisions on requests for funding and gifts in kind. It bases these decisions on charitable criteria, which include:
- Young People
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Unfortunately, we can't say yes to every request we get. So if your application doesn't meet these charitable criteria, we won't be able to accept it.
Any funding decision we make is final. We won't discuss our decisions with applicants.
If you're applying for funding from outside the FirstGroup, you'll need to include a supporting letter from a FirstGroup employee. We'll need this from you at the same time as we receive your application, or we won't be able to consider it.
Off the Buses Charity Newsletter
Off The Buses is our Charity & Sponsorship Committee newsletter in the North Region. You'll find the latest issue here.
Community Investment Exclusion Policy
We also have a Community Investment Exclusion Policy, which identifies types of initiatives and causes that we do not fund. These include:
- Political parties or bodies
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How to Apply
We're sorry to advise that we're unable to accept new applications after Friday 11 April 2014.
We hope to provide information on the new arrangements in the near future.


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Friday, February 28, 2014
The Cuthbert Community Center was packed full Monday night as around 40 people attended the Summerville Vision Plan kick-off workshop.
The 6:30 p.m. meeting launched a weeklong design workshop, or charrette, in which community members voiced their opinions on Summervilles future. The topic-basic workshops were designed to foster discussions that will help the contracted planners, the Lawrence Group, construct the towns 20-year Vision Plan.
The Lawrence Group has a variety of planning professionals, including landscape architects, economic planners and urban designers, who will be using the input to create a master plan for the town, which will be presented in late summer 2014.
The Town has never planned beyond a town-wide comprehensive level before, said Monica Holmes, project manager, at the kick-off. She explained to the crowd that the Vision Plan will include small area planning, for focus areas such as Oakbrook, Downtown and the new I-26 interchange, as well as site-level planning, which will draw attention to small details.
This is a critically important meeting, Mayor Bill Collins said. He asked participants to encourage participation from others.
The meetings were widely-attended, Holmes said.
The kick-off presented an opportunity to share national statistics that will affect the growth of Summerville.
Specifically, Holmes mentioned 80 percent of new population growth will be households without children. The trend can be seen locally, as she said more than 60 percent of those who participated in the charrette survey said they wanted more maintenance-free living in downtown Summerville, such as condos and apartments.
The surveys also indicated citizens are looking for more fine dining and entertainment venues, improved streetscaping, a new mixed-use town center and destinations for ecotourism.
The Cuthbert Center, which the Lawrence Group has taken over for the week, is filled with design boards, sketches, maps, workshop notes and diagrams.
The kick-off was a microcosm of the rest of the weeks events. Discussion took place on five topics: place making and town centers, housing, open spaces, transportation and design guidelines and regulations. Those topics were further broken down during the remainder of the weeks workshops.
At the conclusion of each day, the Lawrence Group held a project update reflecting on the days progress. The charrette will culminate in a closing presentation, which will be given at the Town Hall council chambers on March 6 at 6:30 p.m.
If a person was unable to attend the charrette but would like to contribute ideas, they may fill out the online survey at www.summerville.sc.us/visionplan
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Friday, June 29, 2012
The Georgetown County Sheriffs Office has been asked to investigate a fire that destroyed a mobile home in the 200 block of Meadow Street in the Graves Station community.
The fire was reported at about 9:30 p.m. and was fully involved when firefighters arrived.
The fire is considered suspicious because lo one was living in the mobile home and the electricity had been disconnected.
South Strand News is pleased to offer readers the enhanced ability to comment on stories. We expect our readers to engage in lively, yet civil discourse. We do not edit user submitted statements and we cannot promise that readers will not occasionally find offensive or inaccurate comments posted in the comments area. Responsibility for the statements posted lies with the person submitting the comment, not South Strand News.
If you find a comment that is objectionable, please click "report abuse" and we will review it for possible removal. Please be reminded, however, that in accordance with our Terms of Use and federal law, we are under no obligation to remove any third party comments posted on our website. Read our full terms and conditions.
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This is a unique book that helps its readers put the major (and minor) events in the history of mathematics into the context of what else was happening in the world. Suzuki even shows us that many historical figures in mathematics were actors on a larger stage. Not only is the book a worthwhile read, but it will be a constant reference afterwards. —Victor Katz, Professor of Mathematics Emeritus, University of the District of Columbia, Author of A History of Mathematics
What would Newton see if he looked out his bedroom window? Mathematics in Historical Context describes the world around the important mathematicians of the past, and explores the complex interaction between mathematics, mathematicians, and society. It takes the reader on a grand tour of history from the ancient Egyptians to the twentieth century to show how mathematicians and mathematics were affected by the outside world, and at the same time how the outside world was affected by mathematics and mathematicians. Part biography, part mathematics, and part history, this book provides the interested layperson the background to understand mathematics and the history of mathematics, and is suitable for supplemental reading in any history of mathematics course.
Mathematics in Historical Context provides historical background to the development of mathematics. It can be used as a supplemental text to a history of mathematics course, or even as a stand-alone history text.
Electronic ISBN: 9781614445029
|Mathematics in Historical Context


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Polyblends India Pvt. Ltd.
About us | Products | Services | Facilities | Quality | Specialization | Contact Us |
PBI was established in 1996 as small unit for the manufacturer of master batches. After establishing its name and credentials, the company diversified in the field of specialty engineering plastics. Presently, 15mt/ day of different grades of both master batches & Compounds are being manufacture under strict quality control. This is achieved using indigenously developed technology, modem process control devices monitored by microprocessor and backed by a competent team of technical personal to ensure strict quality norms. Today, PBIs real strength lies in catering tailor made compound for various applications which are being approved and patronized by numerous quality conscious customers all over the country.
Mfrs. & Suppliers of Masterbatches, Polymin(Filled Compounds), Polycol(Colour Masterbatches), Polybatch(Additive Masterbatches)
The production facilities of Polyblends group are located New Delhi, Manesar [gurgaon] & Noida [u.p] . The company has three factories equipped with twin screw extruders, kneaders, mixers and latest microprocessor injection moulding machine, well equipped labs for testing and spectrophotometers for colour matching. With a team of engineers and plastics technologists overseeing and supervising the production process, the company has a capacity to manufacture 15mt/day of engineering plastics & masterbatches.
The company has effective marketing networks all over the country & reputation as a manufacturer of top quality master batches and compounds. The company is certified ISO 9001-2000.
The company is equipped with all the latest & sophisticated testing machines to test the Raw Material and Finished Goods.
POLYMIN series of POLYBLENDS comprise of filles in Polypropylene, Nylon-6, Nylon-66, Polybutylene Terephthalate(PBT), Acrylonitryle butadience styrene(ABS), Polystyrene(PS), Polycaronate(PC) etc.
About us | Products | Services | Facilities | Quality | Specialization | Contact Us |


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Ladbrokes Promo Code
Among the largest and greatest bookmaker in the UK is Ladbrokes, which was established in 1886. Based in Rayners Lane, Ladbrokes was named Hilton Group plc for a brief period of time when it had the hotel brand Hilton under its possession. Ladbrokes made it to the London Stock Exchange with a membership of FTSE 250 Index after being relegated from the FTSE 100 Index during June of 2006.
Ladbrokes Promo Code 2015
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More about Ladbrokes
Initially, the clientele of Ladbrokes consisted mainly of the British elite class and its aristocrats until its acquisition by Cyril Stein and his uncle Mark Stein in 1956. Under the chairmanship of Cyril Stein, Ladbrokes acquired Texas Homecare in 1986 and Vernons Football Pools in the year 1989.
After the Gambling Act of 2005 was introduced in the UK, it subsequently relaxed the laws of advertising for all the gambling companies.
However, when Ladbrokes launched a TV campaign in 2007 that included a group of ex-footballers, it resulted in the very first complaint that was directed to the Advertising Standards Authority (ASA). Nevertheless, it was cleared by ASA. Around this time, SportTech bought the brand Vernons!
A Brief History of Ladbrokes
Over the course of the past decade, Ladbrokes has acquired a huge market position worldwide. Ladbrokes generates employment for more than 14,000 people in the UK alone with a total of more than 16,000 employees worldwide.
Ladbrokes has acquired a leading position in the market of retail bookmaking all over Ireland, UK, Spain and Belgium where there are over 2,700 betting outlets. Ladbrokes also facilitates betting at nine racecourses (which include Ascot) and at eight grounds of the FA Premiership. There is a promo code that can be used to get a free bet on the football, or free casino chips. This can be found near the top of this screen.
Apart from its vast retail presence, Ladbrokes also offers a considerable number of markets in betting daily which can be accessed through the internet, telephone as well as your mobiles. Telephone betting serves around 85,000 customers, while it has drawn in the active participation of almost 800,000 clients.
Ladbrokes offers betting through 13 sites available in nine languages, which also supports 17 currencies. The site provides security of the highest level. So, you're basically in safe hands!
All Round Customer Service
The customer support of Ladbrokes has a reputation of being efficient because it offers its services for 24 hours, 7 days a week through both, email and telephone. Though the option of live chat is unavailable, Ladbrokes offers services in multiple languages like Spanish, English, Swedish, German, Italian, and many others.
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Deposits can be made through several methods like debit and credit cards, using cash at their betting outlets, AstroPayCard, PayPal, SmartPay, Western Union, Neteller, Skrill (MoneyBookers) and many more. The deposits are free of charge and instant. The processing time of the deposits made via bank transfers and Western Union is generally up to five business days. The minimum deposit amount is between £5 and £10, depending on the deposit option you've chosen.
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Overall, Ladbrokes is a great site for sports betting and playing the casino games. So, come experience the fun of entertainment and making money! To get more bang for your buck, make sure to check out the Ladbrokes promo code at the top of this screen.


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Session D2: Topological Surface States
2:30 PM–5:30 PM, Monday, March 21, 2011
Room: Ballroom A2
Sponsoring Unit: DCMP
Chair: Ali Yazdani, Princeton University
Abstract ID: BAPS.2011.MAR.D2.1
Abstract: D2.00001 : Visualizing surface states of topological insulators using spectroscopic mapping with the scanning tunneling microscope
Preview Abstract MathJax On | Off Abstract
Pedram Roushan
(Princeton University)
In topological insulators, the spin texture of the surface states makes them distinct from conventional two-dimensional electron states, and leads to novel properties for these states. These surface states are expected to be immune to localization and to overcome barriers caused by material imperfections. We have used scanning tunneling microscopy and spectroscopy to study the topological surface states in \textit{Bi}$_{0.9}$\textit{Sb}$_{0.1}$\textit{, Sb, } and \textit{Bi}$_{2}$\textit{Te}$_{3}$. By mapping the interference of the surface states scattering off random alloying disorder in \textit{Bi}$_{0.9}$\textit{Sb}$_{0.1}$, we have demonstrated that despite strong atomic scale disorder, backscattering between states of opposite momentum and opposite spin is absent, resulting from the spin texture [1]. Furthermore, we have measured the transmission and reflection of topological surface states of \textit{Sb} through atomic terraces [2]. In contrast to Schottky surface states of noble metals, these surface states penetrate such barriers with high probability. To examine the possibility of disorder induced localization, we investigated the surface states of Bi$_{2}$Te$_{3}$ in the presence of local defects. In the presence of magnetic dopants, we have observed an interference pattern throughout a broad range of energies, even in the region of linear dispersion near the Dirac point [3]. We discuss the results of a statistical analysis of these patterns which can help to learn about the tendency toward localization for these surface states and how this trend is affected as the energy is tuned to the Dirac point. *Work was done in collaboration with J. Seo, H. Beidenkopf, L. Gorman, Y. S. Hor, C. Parker, D. Hsieh, and A. Richardella, M. Z. Hasan, R. Cava, and A. Yazdani. \\[4pt] [1] P. Roushan \textit{et al.} Nature 460, 1106 (2009). \\[0pt] [2] J. Seo \textit{et al.} Nature 466, 343 (2010). \\[0pt] [3] H. Beidenkopf \textit{et al.} (2010).
To cite this abstract, use the following reference: http://meetings.aps.org/link/BAPS.2011.MAR.D2.1


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By Jim Fawcett, ISU Extension field agronomist
Certified crop advisers (CCAs) can earn five hours of credit (three hours in soil and water management, 1.5 in pest management, and 0.5 in crop management) by attending a special CCA morning session, that proceeds the afternoon spring field day tour at the Southeast Iowa Research and Demonstration Farm near Crawfordsville on June 15. The morning session will begin at 9 a.m., with a presentation by Greg Brenneman, ISU Extension agricultural engineer. Brenneman will cover recent tile drainage research results and 20 years of conservation tillage research. The morning session also will include a presentation on the biology and control of nematodes that feed on corn made by Greg Tylka, ISU Extension plant pathologist. The morning will conclude with a discussion in the field by Jeremy Singer, National Laboratory for Agriculture and the Environment agronomist, who will talk about everything you ever wanted to know about cover crops, but were afraid to ask.
The afternoon tour topics will include: Crop Season Review and Marketing Grain in Crazy Times by Kevin Van Dee, Farm Superintendent and Jim Jensen, ISU Extension farm management specialist; Corn Nematode Management, by Greg Tylka; Cover Crop Establishment and Management, by Jeremy Singer; and Making Fungicide Application Decisions, by Mark Carlton, ISU Extension field agronomist. Tours will begin at 1 p.m.Registration for CCAs will begin at 8:30 a.m. The registration fee is $50, which includes lunch. Please pre-register by calling the Johnson County Extension Office at (319) 337-2145, or send an email note to Jim Fawcett at email@example.com by June 13 to avoid a $20 late fee. The registration fee can be paid at the door. To reach the research farm go 1.75 miles south of Crawfordsville on Highway 218, then 2 miles east on G-62, then 0.75 mile north.
Jim Fawcett is an Iowa State University Extension field agronomist serving eastern Iowa. He can be reached at 319-337-2145 or by emailing email@example.com.
This article was published originally on 5/25/2011 The information contained within the article may or may not be up to date depending on when you are accessing the information.
Links to this material are strongly encouraged. This article may be republished without further permission if it is published as written and includes credit to the author, Integrated Crop Management News and Iowa State University Extension. Prior permission from the author is required if this article is republished in any other manner.
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text:
Systems and methods for facilitating electronic transactions via a device capable of communicating with a network. The method includes providing a catalog on the network and communicating with the network via the device. The catalog is downloaded to the device and communication is discontinued with the network via the device. At least one desired transaction within the catalog is selected and stored on the device. The device recommunicates with the network and the at least one desired transaction is completed.
1. A method for facilitating electronic transactions via a device capable of communicating with a network, the method comprising:
providing a server having a catalog of assets and a plurality of assets;
providing the catalog of assets from the server to the network;
communicating with the network a first time via the device;
providing the catalog to the device from the network;
discontinuing the first time communication with the network via the device;
when the device is not in communication with the network, selecting at least one desired transaction within the catalog, the at least one desired transaction including selecting an asset from the catalog, which selected asset can be downloaded to the device;
storing the at least one desired transaction on the device;
communicating with the network a second time via the device in response to the selection of the transaction;
completing the at least one desired transaction, the at least one desired transaction comprising:
retrieving the selected asset from the server with the plurality of assets;
providing the selected asset from the server to the network; and
downloading the selected asset to the device from the network;
discontinuing the second time communication with the network via the device;
modifying the selected asset on the device;
storing the modifications of the selected asset to the device;
communicating with the network a third time via the device; and
uploading the stored modifications to the network.
2. A method in accordance with
3. A method in accordance with
4. A method in accordance with
5. A method in accordance with
6. A method in accordance with
requesting an update of the catalog on the device from the network;
identifying changes made since a previous catalog update;
comparing a last update date and time for the catalog on the device to a list of changes made to the catalog on the network to determine items that need to be provided to the device in the update;
downloading from the network to the device only the items; and
updating the catalog on the device with the items.
7. A method in accordance with
8. A method in accordance with
9. A method in accordance with
10. A method in accordance with
11. A method in accordance with
12. A method in accordance with
13. A method in accordance with
14. A method in accordance with
15. A method in accordance with
16. An electronic transactions system comprising:
a server having a catalog of assets and a plurality of assets;
a network in communication with the server;
a device capable of communicating with the network to download the catalog from the server to the device;
wherein a desired transaction is indicated on the catalog when the device is not in communication with the network, the at least one desired transaction is further indicated by selecting an asset from the catalog, which selected asset can be downloaded to the device;
wherein the desired transaction is completed, in response to the selection of the asset, by retrieving the selected asset from the server with the plurality of assets, providing the selected asset from the server to the network, and downloading the selected asset to the device when the device subsequently communicates with the network;
wherein the selected asset is modified on the device;
wherein the modifications of the selected asset are stored to the device; and
wherein the stored modifications are uploaded to the network.
17. An electronic transactions system in accordance with
18. An electronic transactions system in accordance with
19. An electronic transactions system in accordance with
20. An electronic transactions system comprising:
means for providing a server having a centralized catalog of assets and a plurality of assets;
means for providing the centralized catalog of assets and the plurality of assets from the server;
means for reading textual content and being capable of communicating with the means for providing the centralized catalog of assets a first time;
means for downloading a catalog from the means for providing the centralized catalog of assets and the plurality of assets from the server to the means for reading textual content;
means for selecting a desired transaction from the catalog at the means for reading textual content when the means for reading textual content is not communicating with means for providing the centralized catalog and the plurality of assets from the server the first time, the means for selecting a desired transaction including means for selecting an asset from the catalog, which selected asset can be downloaded to the device;
means for completing the desired transaction, in response to the selection of the transaction, by retrieving the selected asset from the server with the plurality of assets, providing the selected asset from the server to the means for providing the plurality of assets, and downloading the selected asset when the means for reading textual content communicates with the means for providing the centralized catalog and the plurality of assets from the server a second time;
means for modifying the selected asset at the means for reading textual content when the means for reading textual content is not communicating with the means for providing a centralized catalog the second time;
means for storing the modifications of the selected asset at the means for reading textual content; and
means for uploading the stored modifications to the means for providing the centralized catalog and the plurality of assets from the server when the means for reading textual content communicates with the means for providing the centralized catalog and the plurality of assets from the server a third time.
This application claims priority from U.S. Provisional Application No. 60/280,460 filed Mar. 30, 2001; No. 60/280,461 filed Mar. 30, 2001; No. 60/280,459 filed Mar. 30, 2001; No. 60/280,458 filed Mar. 30, 2001 and No. 60/280,456 filed Mar. 30, 2001, which are all incorporated herein in their entireties by reference purposes.
Not Applicable
Not Applicable
1. Field of the Invention
The present invention relates to systems and methods for an electronic off-line catalog, and more particularly, to a catalog of electronic books, the catalog may be downloaded to an electronic device and then accessed while the electronic display device is off-line, with transactions, such as purchases, being fulfilled and updates occurring when the electronic device is back on line.
2. Description of the Prior Art
Many items are currently available for purchase over networks such as the internet. Generally, when one wishes to purchase items over the internet, one goes to a website or location that generally includes a catalog. The catalog is viewed and selections are made while the person is on-line and at the website. Typically, on-line catalogs are only available for PCs or other platforms with browser-based environments.
If the catalog is large and has many choices, it may be time consuming to browse through the catalog. This could mean tying up one's internet connection, often a telephone line.
Furthermore, on-line catalogs may or may not change frequently. Often, even when the on-line catalog changes, the changes are minor.
Accordingly, it is desirable to have access to a catalog when one is actually off-line. This would allow a user to peruse the catalog at their leisure without tying up an internet connection and/or telephone line. Additionally, the user would have access to the catalog even if access to the internet is not available.
The present invention provides a method for facilitating electronic transactions via a device capable of communicating with a network. The method includes providing a catalog on the network and communicating with the network via the device. The catalog is downloaded to the device and the network connection is terminated. At least one desired transaction within the catalog is selected (e.g. an item is marked for purchase) and the transaction stored on the device. The device recommunicates with the network and at least one desired transaction is completed (e.g. a purchase is completed, or a purchased item is downloaded to the device).
In accordance with one aspect of the present invention, multiple desired transactions are selected.
In accordance with another aspect of the present invention, the catalog includes multiple sections and a user selects which sections to download, add or update.
In accordance with a further aspect of the present invention, the catalog is updated when the device recommunicates with the network.
In accordance with yet another aspect of the present invention, updating the catalog includes the device requesting a catalog update from a version of the catalog stored on the network. All changes made to the network catalog since the last time the catalog on the device was updated are identified by the network and downloaded to the device. Only the updated content is downloaded from the network and the version of the catalog on the device is updated to create an up to date version of the catalog on the device.
In accordance with a further aspect of the present invention, the updating occurs automatically.
In accordance with another aspect of the present invention, the updating occurs aperiodically.
In accordance with yet another aspect of the present invention, the device recommunicates with the network automatically.
In accordance with a further aspect of the present invention, the device recommunicates with the network manually due to a user request or user interaction.
The present invention also provides an electronic transactions system that includes a network, a device capable of communicating with the network, and a catalog representing a plurality of possible transactions. The catalog is downloaded to the device from the network. A desired transaction is indicated using the catalog when the device is not in communication with the network. The desired transaction is completed when the device subsequently communicates with the network.
In accordance with one aspect of the present invention, the device is an electronic book.
Accordingly, the present invention provides systems and methods for an electronic catalog that may be obtained from a network and viewed while off line with the network. Transactions may be initiated off-line with the catalog and are completed when back on line with the network. The present invention allows for the catalog to be customized to a user's preferences and may be updated when communication with the network is reestablished. The off-line catalog in accordance with the present invention is especially suited for stand-alone devices.
Other features and advantages of the present invention will be understood upon reading and understanding the description of the preferred exemplary embodiments, found hereinbelow, in conjunction with reference to the drawings, in which like numerals represent like elements.
The present invention is directed to a catalog that is downloadable from a network such as, for example, the internet. It is especially suitable for electronic books, and systems and methods related thereto that are described, for example, in the present assignee's copending patent application Ser. No. 09/419,554, and issued U.S. Pat. No. 6,275,934, both of which are hereby incorporated herein in their entireties for all purposes. While for clarity, the present invention will be described with reference to electronic books, and systems and methods related thereto, those skilled in the art will understand that the present invention may be used for various other applications, and hence should not be unduly limited.
Portable Electronic Devices Such As E-Book Readers
Referring to
System 100 preferably includes more than one portable electronic book 10, to be commercially viable. This is illustrated in
In one embodiment of the invention, system 100 further comprises a secondary virtual bookstore 60 in electrical communication with the information services system 20. In this case, the information services system 20 also includes a directory of virtual bookstores 26 in order to provide the portable electronic book 10 with access to the secondary virtual bookstore 60 and its catalog of digital contents.
The information services system 20 also includes a registration server 24 for keeping track of the portable electronic books that are considered active accounts in the system and for ensuring that each portable electronic book is associated with a primary virtual bookstore in the system.
The information services system 20 preferably includes a local bookshelf 30 associated with each portable electronic book 10 in the system. Each local bookshelf 30 contains all digital contents requested and owned by the associated portable electronic book 10. Each portable electronic book 10 user may permanently delete any of the owned digital contents from the associated local bookshelf 30. Since the local bookshelf 30 contains all the digital contents owned by the associated portable electronic book 10, these digital contents may have originated from different virtual bookstores. The local bookshelf 30 is a storage extension for the portable electronic book 10. Such storage extension is needed since the portable electronic book 10 generally has limited non-volatile memory capacity.
The user of the portable electronic book 10 may add marks, such as bookmarks, inking, highlighting and underlining, and annotations on a digital content displayed on the screen of the portable electronic book, then stores this marked digital content in the nonvolatile memory of the electronic book 10. The user may also upload this marked digital content to the information services system 20 to store it in the centralized bookshelf 30 associated with the portable electronic book 10, for later retrieval. It is noted that there is no need to upload any unmarked digital content, since it was already stored in the local bookshelf 30 at the time it was first requested by the portable electronic book 10.
The information services system 20 further includes an Internet Services Provider (ISP) 34 for providing Internet network access to each portable electronic book in the system.
The housing 210 provides overall housing structure for the electronic book. This includes the housing for the electronic subsystems, circuits, and components of the overall system. The electronic book is intended for portable use; therefore, the power supply is mainly from batteries. The battery holder 215 is attached to the housing 210 at the spine of the electronic book 10. Other power sources such as AC power can also be derived from interface circuits located in the battery holder 215. The cover 220 is usually made by flexible material and is attached to the housing 210. The cover is used to protect the viewing area 230.
The display screen 230 provides a viewing area for the user to view the electronic reading materials retrieved from the storage devices or downloaded from the communication network. The display screen 230 may be sufficiently lit so that the user can read without the aid of other light sources. When the electronic book is in use, the user interacts with the electronic book via a soft menu 232. The soft menu 232 displays icons allowing the user to select functions. Examples of these functional icons include go, views, search, pens, bookmarks, markups, and close. Each of these icons may also include additional items. These additional items are displayed in a drop-down tray when the corresponding functional icon or key is activated by the user. An example of a drop-down tray is the pens tray which includes additional items such as pen, highlighter, and eraser. In one embodiment, the soft menu 232 may be updated dynamically and remotely via the communication network.
The page turning mechanism 240 provides a means to turn the page either backward or forward. The page turning mechanism 240 may be implemented by a mechanical element with a rotary action. When the element is rotated in one direction, the electronic book will turn the pages in one direction. When the element is turned in the opposite direction, the electronic book will also turn in the opposite direction. The page turner mechanism 240 may also be used as a latch to hold the cover 220 in place when the electronic book is closed.
The menu key 250 is used to activate the soft menu 232 and to select the functional icons. The bookshelf key 255 is used to display the contents stored in the bookshelf and to activate other bookshelf functions. The functional key 254 is used for other functions.
The electronic book 10 includes a view switching feature that allows readers or users to increase or decrease the size of the font used to create page display images to suit the preferences of the readers or users. As stated previously, a page display image is an arrangement of pixels on a display screen or an output device to create a visual representation of a page of reading material. Each set of page display images of an electronic publication, document, or reading material that is generated using a set of view parameters is referred to as a page display view. View parameters may include the point size of the font that should be used to create page display images. View parameters may also include the dimensions of a display screen or a portion of a display screen of the electronic book where page display images are presented.
Electronic book 10 preferably supports two different page display views such that page display images of the first view are generated using a smaller font than page display images of the second view. In practice, the electronic book 10 can be constructed to support more than two different page display views.
Catalog Definitions
Catalog—The document that users download, through which they can order content.
Catalog Section—The Catalog is comprised of a number of Sections, e.g., table of contents, a "Featured" section, etc.
Asset—Something for sale. Catalog sections contain descriptions of which assets they offer for sale.
Template—Something that specifies the "look" of a particular portion of a catalog—a section header, an asset listing, etc.
Catalog Image—Catalogs have a set of images, images that are created especially for catalogs.
Catalog Creation
The "Structure" of a Catalog is the data associated with a catalog, without regard to how that data is presented visually: what sections are in the catalog, in what order, what the catalog should be called, etc. The Catalog structure is similar to a "tree." At the root of the tree is the "Catalog" item. Below that, there are two kinds of items: items which specify the "top-level" information about the catalog, for example, its name, and a set of Catalog sections. Each of those sections has two types of data associated with it: data that describes the Section itself (its name, etc.) and then the set of Catalog assets that are listed in that section. Finally, each asset may itself have various pieces of information associated with it how it should display itself, etc.
Preferably, each catalog includes a cover page and a Table of Contents (which might be multiple pages).
Preferably, a number of basic catalog "styles" are provided within the catalog, such as, for example, promotional features, index listings and periodical layouts. Combinations of these styles with a particular focus are used to create the final catalog.
The promotional featured items are particular assets selected from a database of available assets to be included in a "features" section. These featured assets may either be selected manually or automatically. For manual selection, a catalog creator may specify which assets are to be featured assets and the order in which they are to appear. Featured asset information may include, for example, the title, author, custom catalog description, thumbnail of the cover jacket or other photo artwork, publisher, publication date and e-book publish date, the current list price, the electronic book provider's price, and the savings if any. If there are no savings, the savings item will not appear at all. An example of a promotional feature listing is illustrated in
For implementation, styles are preferably mapped into templates. In order to maximize flexibility and screen space utilization the Featured Title style may, in fact, map to several templates based on the size of the content item. For example, Featured Titles may consist of 3 template choices: "Full Page layout", "� page layout", and "minimal layout".
The index listing format is an abbreviated summary of assets (titles) to maximize the number of items that may be included in a limited storage space. For this reason, index items preferably include, for example, the title, author, publisher, abbreviated or short description, list price, and the electronic book provider's price. An example of the index listing format is illustrated in
The periodical style is specific to periodicals, newspapers or magazines. A sample listing is illustrated in
As can be seen in the Figures, the styles are all preferably presented with an Order button that is visually similar in style to the current button options on an reading device. Items that have already been requested and added to an order may be indicated by changing the button label to "ordered". If the user taps the ordered button they should be taken to the order form showing the page on which that item is listed. "Back to Catalog" is preferably provided as a hot link to return them. If an item has not only been ordered, but the order form has been processed, then that item's button label may be set to "purchased."
Other sections may include "Bestsellers." The default is to include all books with the asset type of "bestseller" in the catalog database; however, the catalog author may alter the number of books included. "What's New" may be included and includes book and possibly periodicals that have most recently been added to the bookstore across all topic areas (See
Thus, a single catalog is available for an on-line bookstore. This single catalog is formatted with a number of sections, reflected in the Table of Contents of that particular catalog. The sections present different subsets of assets available by providing the user with different views. This is a single dynamic data set that is presented in differing ways (i.e., these sections are views or templates on a single database so there is maximal space efficiency). From the user's perspective this will appear as a single electronic book with multiple sections in that book. Assets may appear in multiple sections but in a different ordering and presentation. In a preferred embodiment, even though an asset may appear in multiple sections, the data describing the asset should only be stored once in the underlying data set as will be more fully explained herein.
Catalog Content
A catalog will generally have a maximum size for obvious reasons (e.g. storage constraints on the device, or network bandwidth constraints on catalog download). In order to ensure that the most useful information is included, catalogs will eliminate the least popular items when they exceed the maximum permitted size. In particular, items will be included according to the following criteria: Features—Items marked by the catalog creator/author to be featured in that catalog for a particular time frame will automatically be guaranteed a place; Most recent—Items that have become available most recently in time based on the current date; Most sales—Items that have generated the largest volume of sales since their availability will be included; Not already purchased by the user; and, Some items may be offered at certain prices for "limited time offers". In this case an "expiration date" can be set for that item to be featured or for a particular price to be included. Once expired, an item can be automatically removed from the feature list and/or an item price can be automatically reset. Those skilled in the art will understand that the above described process and criteria are merely exemplary and other variants may be used.
The intent is to set a maximum number of items to be included, for example 5000. To determine the items to be included some percentage will be allocated to most recently available items. This will be independent of the sales figures for these items since new acquisitions will initially have 0 sales and may take several days or even weeks to attain significant sales volumes. Secondly, the remaining percentage of the total will be allocated to the items with the highest total sales volume, which will be used as a rough measure of popularity.
On a per user basis, items that a particular user has already purchased may be eliminated from the catalog listings to maximize visibility of new items for that particular person.
With the present invention, inclusion criteria are used so that an asset is only downloaded or transmitted to the reading device once, even if it appears multiple times in the catalog. For each section, the network looks at the inclusion criteria to see which assets go into that section. These assets are all pooled into an internal data set, so that even if an asset is in eight different sections, it only occupies storage once in the data set, but with an annotation that indicates which eight sections requested it.
Once the network knows the set of assets, it looks to see which images are necessary. Only a small subset of the asset images are sent (e.g., only those images that are referenced by featured or bestsellers assets, identified by templates, are included). This helps minimize the size of the download.
New Users
Preferably, purchasers of a reading device will automatically receive a catalog by default. They can then choose to continue with their subscription, customize their catalog, stop their subscription (i.e., block future updates), or remove the catalog from their device's local bookshelf altogether.
Initially when users receive a brand new reading device, they will preferably receive a hidden catalog. The catalog will be loaded on the device but preferably will not initially be accessible or visible to the user. Since reading devices can remain in inventory for some period of time it is possible or even likely that this catalog will be out of date or the pricing information may have been modified. Downloading the entire catalog rather than merely updating the items that have changed is extremely time consuming and would require an extended network connection, which is unacceptable from a user experience perspective. Therefore, it is preferable to initially load the catalog when the device receives its original software during manufacturing, but mark the catalog as "hidden" or invisible content until the user registers the device, i.e., the device is purchased and activated. At this time the catalog may be updated based on the latest available information and it will be activated and hence visible. This will ensure a minimal amount of connect time while guaranteeing that the users will see accurate pricing and title information in catalogs provided to them when they first purchase a reading device.
In practice the following steps will occur. Upon registration—which preferably will always occur automatically the very first time the user connects to a network—users will be asked to enter their registration information. Following this, users will see a message indicating that they are being registered, followed by a message indicating that they are receiving our latest catalog of available titles, special offers, etc. During this brief time interval, the database is updated on the local device with any information that has changed since the device received its software. When registration and catalog updating are completed, the user sees a new category item called "Catalogs" on their local bookshelf with the new catalog. (See
Updating and Customizing the Catalog
Preferably, a user may either automatically receive the catalog from the network or may receive it upon request from the online bookstore. Preferably, the off-line catalog may not have its subscription canceled. If it exists on the local bookshelf, then the updates will occur whenever a connection is made. Alternatively, users may opt to remove it from their reading device (preferably with a warning message). It will still remain on the online bookshelf where it cannot be removed. If re-requested by the user, when downloaded the latest information updates will also be retrieved from the bookstore.
Preferably, to generate new content for catalogs, the bookstore administrator may access the bookstore and modify the existing catalog and save it. These "updates" are preferably done automatically when the reader communicates with the server. Updating is preferably done by comparing the most recent update date of the catalog on the device with the update date of each piece of information on the server. Only those portions of the catalog on the server that are different are then downloaded to replace the corresponding portions of the catalog on the reader.
When a connection to the network occurs for a catalog update, the reading device relays (a) which catalog it wants, (b) which catalog sections it wants, and (c) how old its existing catalog is with a time stamp parameter. The network then looks at a Catalog Item table, which includes all catalog items arranged chronologically, and the network sends all the items that are newer than the existing time stamp for the existing catalog.
As an example, assume there are 300 items that need to be sent, and the device can only handle 50 in a single "chunk." This means that if the current time stamp is S, then the network needs to send a new time stamp value of "S+50." However, the value of the new time stamp comes at the top of the packet that the network sends to the device. This means that the network needs to do two passes through the Catalog Item table—one to figure out what the next time stamp will be and one to send everything up to that time stamp.
Because of selective section inclusion, this is now a two-segment process. Suppose that a device connects with a catalog that is 2 weeks old, and they want 3 new sections for the first time ever. Before giving them a "here is what's new from the last 2 weeks" update, the network needs to give them a "here are the 3 new sections" update.
There are considerations in addition to the time stamp in selecting the information to transmit and order of updates. Consider the following: many items may share the same time stamp. For example, all assets that have never changed may have a time stamp of Jan. 1, 1970. A "tie-breaker" needs to be provided. Preferably, there are two levels of tiebreaker. Firstly, there is a "priority", with higher priority items being sent first. For example, a section definition needs to be sent before any attributes for that section are sent. Secondly, within that, the ID of the Catalog Item in the database needs to be sent.
Sometimes it may be desirable to ignore the existing time stamp, to just send everything and do a complete download. This is mainly done when debugging the off-line catalog. Therefore, one of the attributes of a time stamp is a "cliff." If a time stamp comes in and it has a "cliff" that is less than the current "cliff" for that catalog, then the time stamp is ignored, and a complete download is done.
Updating may be a two-pass process, because of catalog selective inclusion and exclusion. That means that the time stamp really needs to contain two "mini time stamps" one to indicate the processing pass, and one to indicate progress within that pass.
Customizing a catalog is preferably accomplished by navigating to the Table of Contents section of the catalog and selecting the desired sections of the catalog by checking or un-checking the desired sections to include or exclude from the catalog. In this way, users may add and delete, for example, sections that will be included in their off-line catalog.
User customizations to the catalog are preferably done on the local catalog version that exists on the local bookshelf on the reader, i.e., the off-line version. The on-line bookshelf version of the catalog remains unchanged. Users may easily get back to the original catalog by pressing the download catalog option (in addition to also being able to simply reset their customization to include all content types).
Thus, with reference to
Secure Transactions
Several possible combinations of security and credit card information are possible, at the discretion of the user. Security is based on whether or not the user has decided to store their credit card information on the reading device to facilitate future purchases. If the user elects not to do this, they will be prompted to enter new credit card data for each session into a blank form associated with the order form before they will be authorized to order any items. If a user decides to save credit card information so that they do not have to re-enter it each time, preferably they will enable and set a passcode. Entering the passcode will act as authorization for the purchase. It will be requested once during each session (the first time the order/buy button is pressed). However, it is possible for users to save credit card information and to not set any passcode. In this case, any order requests will be added to the order form and purchased without further authentication from the user (beyond displaying normal status messages).
Credit card information handling has a number of aspects impacting security and user experience. For new users, the very first time they try to buy through the off-line catalog, their credit card information will be blank. In this case, when the user first presses the order button (or buy button if online) they will be prompted for this information since they have no credit card information on file. A form will appear (See
Once entered, the user's credit card information may be encrypted and, if enabled, saved on the device. Any problems validating the credit card information preferably will be reported to the user via a message tray the next time the user powers on the device. Credit card data may be re-accessed and modified from the "Secure Credit Card Information" form presented as the last page of the Order Form "book." When displayed, the existing credit card number will show only the last 4 digits for additional security.
Users may change designated credit cards for the order purchase before it occurs by paging to the "Secure Credit Card Information" form presented as the last page of the "Order Form" book.
For an order form to be useful for completing a purchase, credit card information must be attached (even if not saved for future purchases). Users enter the new credit card information that will apply to that as-yet-unprocessed order. If the user does not check the "save" option, the new credit card will only apply to that particular order form. Any items ordered subsequently as part of that same session (prior to the order being processed) will reflect the new credit card information as part of the confirmation message tray and will be added to that same order form. Once the order has been processed, only saved credit card information will apply to future orders. If the user does check the "save" option then this new credit card information will apply both to the current order form and to any future orders. The new information will replace the old credit card information, if any.
Preferably, order forms always exist on the local bookshelf even when no items have been requested, in which case they are blank. Orders are generated automatically whenever the user taps on the "order" button next to a title or periodical item. The order form preferably appears as a separate document and may be separately viewed and modified, if desired, by the user. Subsequent orders are appended to the order form.
Preferably, all pages of the catalog include a hot link item called "order form." Pressing this item at any time closes the catalog (preferably at the current page location, which is saved for later reference) and opens the order form document. If the user wishes to return to the catalog, they do so by either explicitly selecting the catalog title from the bookshelf or by pressing a hot link in the order form labeled "back to catalog". Either of these two options returns the user to the last page viewed in the catalog.
Order forms preferably are treated in "book-like" ways. The user may select the "get info" icon that will result in a tray describing the status of the order, the number of items and the size of the file. The "get info" tray preferably has the normal book related options: open, remove, etc. Tapping on the order form title will open that order form document for review or changes. Items preferably may be removed at this time. A running total is preferably provided. To add more items to the order, the user preferably taps the "back to catalog" link or opens the catalog from the local bookshelf by tapping on the catalog title.
Order forms may be processed the next time the reader connects (either the user requests a connection or the device automatically connects), or are processed immediately when the user taps "buy now." Once payment information is validated on-line, each of the items ordered is delivered automatically to the user's on-line bookshelf where it preferably appears in the New category. Following this, the server may execute its normal automatic download procedures to download all items in the New category from the online bookshelf to the local bookshelf. The newly purchased content is handled as any other new content from this point on.
Users may choose to purchase the items "now" through the order form if they do not wish to wait, i.e., while on-line. The "buy now" button or icon on the order form will initiate a network connection to the bookstore using whatever means possible and, once connected, will send the order form off to the bookstore for immediate processing. If payment authentication is authorized, the newly purchased items will immediately appear in the online bookshelf.
Accordingly, with reference to
In summary, users browse the catalog with their reading device. This may occur on-line or off-line. Upon deciding to make a purchase, the user makes the appropriate selection and handles payment in accordance with one of the previously described manners. If the device is on-line, then the user indicates "Buy Now" (or the device may automatically handle the purchase). If the device is off-line, then the next time it is on-line—either manually or automatically—the purchase transaction is completed.
Those skilled in the art, will understand that other types of devices may be used for viewing the catalog, such as, for example, computers, televisions (with an appropriate "set-top box"), PDAs (personal digital assistants), etc. Generally, devices that may communicate with a network such as the Internet and that include a display and memory may be used.
Likewise, the catalog could be presented with other forms of electronically deliverable content such as audio, video, etc., in addition to or in place of the text content. The catalog and updates could also be provided non-electronically, for example, with a compact disk, ROM, etc. Also, non-electronically deliverable assets could additionally or alternatively be ordered through the catalog of the present invention, although this would require integration with the necessary fulfillment infrastructure.
The attached Appendix includes examples of protocols for the present invention.
Although the present invention has been described with reference to specific exemplary embodiments, it will be appreciated that it is intended to cover all modifications and equivalents within the scope of the appended claims.
Patent Citations
Non-Patent Citations
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Houston Rodeo
Tales from the Houston Livestock Show and Rodeo
Monthly Archive for March, 2006
The dust has settled
Well, the 2006 Houston Livestock Show and Rodeo is finally done. The last grand entry has made its way around the arena with its legion of waving hands crowded into wagons. The national anthem has been crooned for the last time into its subsequent explosion of fireworks overhead. The calf and goat ropers have tied […] [Read More]
Vegas in town for a spell
Bill Olive : For the Chronicle The rotating tower of Vegas. Every evening at the Houston Livestock Show and Rodeo, when the sun sets and people are still showing up and getting their tickets punched to get into the show, a little bit of Las Vegas turns up. That towering ferris wheel (which I won't […] [Read More]
Everybody wants a ride
You don't have to be a cowboy or cowgirl to get a ride on a bovine in these parts. Heather Reams has brought a herd of Longhorns in from her home in Joshua so that kids can get their pictures taken atop the giant beasts. Why Longhorns? Well, for one, they look really cool when […] [Read More]
Rodeo picks for the weekend
Friday (March 17) • If you haven't yet seen one of the Peruvian Horse Exhibitions, by all means turn up early and see them strut their stuff in Reliant Center. It's quick — only about 30 minutes, but totally worth it if you wanna see horses do all kinds of crazy-leg walking and the like. […] [Read More]
Weighing in
You haven't heard anything until you've heard a pig freaking out. Out from behind a retaining wall in the back of Reliant Center, where folks were washing up their hogs for show, there was a squeal. But this wasn't just a squeal. It was almost a roar — desperate, confused, high-pitched and absolutely bloodcurdling. It […] [Read More]
Go rodeo Tejano
Sunday's Go Tejano Day ended up being one of the most inspiring RodeoHouston performances to date. With an almost Vegas-like opening set by award-winning San Antonio vocalist Jay Perez setting the tone, the yearly mariachi contest followed at the far end of the arena on a tiny stage backed by a tall blue curtain. Femenil […] [Read More]
Bert's best friend(s)
As we stood at the mouth of the tunnel, looking out on the floor that serves as the sort of faux Colosseum that is RodeoHouston, we couldn't help but notice the man walking up dressed all clownish, with suspenders pulled up to his chest and five dogs tied to leashes tucked into the fingers of […] [Read More]
Reliant City
When Hurricane Katrina hit and busloads of evacuees came roaring into town headed for the Dome, weary and weathered from the storm, the entirety of Reliant Park was all of the sudden bursting with life. Those transplanted Louisiana residents became the settlers of a new city in a way (Reliant City, if you will) over […] [Read More]
Like a raging bull
You know that vision you've had in your head since childhood — the one of a raging bull at the other end of the arena, digging one hoof into the sand, lowering its head and readying to charge? Well that look in the bull's eyes is the same one in the eyes of a steer […] [Read More]
Rodeo picks for the weekend
Friday (Mar. 10) • The Llama/Alpaca show is going on all day. Stop in and take a peek at the animals that Jacko keeps as pets. Details: Reliant Center, 8334 Fannin. 832-667-1000. • Good old Texas music maker Tommy Alverson will be in charge of getting the boots moving at the RodeoHouston after-party. If you […] [Read More]
You two are braver than I
Bill Olive : For the Chronicle Jennifer Whiting and John Glaze ready to blast off. It was a giant eyeball suspended between two metal cables tied to giant chopsticks sticking up from the ground. It would swallow up two people in its skeletal metal belly and then, on command of a man at the button, […] [Read More]
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College of Business Career Center Team
|Lee Brown, Assistant Director
Lee has served as Assistant Director of the COB Career Center since 2006. After graduating from ECU's College of Business, he began his career in the automotive industry. Having worked several years in corporate management, Lee brings a business-based approach to the Career Center. Lee directs the operations with a strong focus on the Corporate Outreach/Employer Relations extension but also assists students with resumes, practice interviews, and other career and professional development activities. Lee also holds a Master in Adult Education.
|Susanne Killian, Career Counselor
Susanne comes to ECU from the Raleigh area bringing twenty years of educational and career development experience to her role as Career Counselor in the College of Business. She is a Certified Career Facilitator and a 2011 graduate of NC State University with a Ph.D. in Educational Research. Susanne's main focus is to assist students in exploring their options through self-assessment, networking opportunities, and internships as well as encouraging students to gather information to better equip themselves to achieve their academic, personal and career goals. Her true belief is that it is never too early to start preparing for your future.


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