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US 5597696 A Abstract The present invention relates to conjugates of a cyanine dye and an oligonucleotide. When these conjugates hybridize or bind to a target, a detectable increase in fluorescence intensity or change in fluorescence polarization is observed.
Claims(10) 1. A compound of the formula ##STR2## wherein X is O, S, Se, N-alkyl having from 1-6 carbons or C(CH3)n ;
R1 is alkyl having from 1-6 carbons; R2 is alkyl having from 1-6 carbons; R3 is fused benzene, alkyl having from 1-6 carbons, methoxy or is absent, R4 is alkyl having from 1-6 carbons, methoxy or is absent; Z is an oligonucleotide having from 8 to 50 bases; n is zero or an integer from 1-6; and n1 is an integer from 2 to 10. 2. The compound of claim 1 wherein X is S.
3. The compound of claim 2 wherein n1 is 3.
4. The compound of claim 2 wherein n1 is 6.
5. The compound of claim 1 wherein X is O.
6. The compound of claim 5 wherein n1 is 3.
7. The compound of claim 5 wherein n1 is 6.
8. The compound of claim 1 wherein X is Se.
9. The compound of claim 8 wherein n1 is 3.
10. The compound of claim 8 wherein n1 is 6.
Description The present invention relates to the covalent attachment of thiazole orange and other related labels to oligonucleotides which are utilized in the detection of nucleic acid targets. The detection of single-stranded nucleic acid targets by hybridization to fluorescently labeled probes is of significant interest for the development of improved reagents for molecular diagnostics. Fluorescently labeled oligonucleotides also are useful probes of nucleic acid structure and hybridization at concentrations below those detectable by other non-isotopic analytical solution-phase methods. Morrison, L. E, and Stols, L. M., Biochemistry 32, 3095 (1993). Cyanine dyes such as thiazole orange have demonstrated large fluorescence intensity increases upon binding to double stranded DNA. Makler, M. T., Lee, L. G., and Rectenwald, D. (1987) Cytometry 8, 568-570; Lee, L. G., Chen, C-H., and Chiu, L. A. (1986) Cytometry 7, 508-517; Lee, L. G. and Chen, C-H., U.S. Pat. No. 4,957,870 (Sep. 18, 1990) "Detection of Reticulocytes, RNA, and DNA"; Lee, L. G. and Chen, C-H., U.S. Pat. No. 4,883,867 (Nov. 28, 1989) "Detection of Reticulocytes, RNA, and DNA". This fluorescence intensity enhancement for thiazole orange has been estimated to be as high as 18,000. Glazer, A. N. and Rye, H. S., Nature 359, 859 (1992). Although covalently linked dye-oligonucleotide complexes have been used to configure assays based on fluorescence energy transfer and quenching, direct tethering of a cyanine dye to an oligonucleotide has not been accomplished to date. The present inventors have addressed this discrepancy in the art by directly tethering cyanine dyes to oligonucleotides to produce covalent cyanine dye-oligonucleotide conjugates. When these conjugates hybridize or bind to a target, a fluorescence intensity increase and/or polarization is observed. The various objects, advantages and novel features of the invention will be more readily appreciated from the following detailed description when read in conjunction with the appended drawing figure in which: FIG. 1 is a graphic depiction of the results of a fluorescence polarization assay with a thiazole-orange labeled oligonucleotide and its complement. Oligonucleotides are utilized in a variety of formats to determine the presence or absence of a particular target of interest. In one format, an oligonucleotide is utilized as a probe to detect a target nucleic acid sequence by hybridizing thereto and thus forming a double stranded or partially double stranded product. In another format, an oligonucleotide known as a nucleic acid ligand or aptamer binds a protein or small molecular target by means other than Watson-Crick type nucleotide hybridization, as taught in U.S. Pat. No. 5,270,163. Similarly, compounds which bind to protein or small molecular targets have been produced by linking two or more oligonucleotides of reverse sequence polarity to a connecting compound. These oligonucleotide compounds are referred to as bi-directional nucleic acid ligand compounds and are more completely described in co-pending U.S. patent application Ser. No. 08/252,071, filed May 31, 1994. The present invention relates to the covalent linking of a cyanine dye to an oligonucleotide (oligonucleotide when used herein is intended to include all oligonucleotide containing compounds including those described above). Upon hybridization or binding of this dye-oligonucleotide conjugate to a target, whether nuclei(acid sequence, protein or small molecule, changes in fluorescence may be detected by either steady state intensity or life time measurements. Hybridization or binding of conjugate to target may also be detected by other fluorescence techniques such as anisotropy or energy transfer techniques. Suitable cyanine dyes for use in the present invention include those described in U.S. Pat. No. 4,883,867 and having the following structure: ##STR1## where X is O, S, Se, N-alkyl (having 1-6 carbons) or C(CH3)n ; R1 is alkyl having from 1-6 carbons; R2 is alkyl having from 1-6 carbons; R3 is fused benzene, alkyl (having 1-6 carbons), methoxy or is absent; R4 is alkyl having 1-6 carbons, methoxy or is absent; and Y is a reactive ester such as N-hydroxysuccinimide or pentafluorophenyloxy acid chlorides; and n is zero or an integer from 1-6. Some of the dyes represented by this structure are thiazole orange and thiazole yellow. Suitable linkers or tethers for combining the dye and oligonucleotide include any linking compound which will bind to the dye through an amide bond. Generally, the tethers are hydrocarbon chains of from 2 to 10 carbons in length which are commercially available from companies such as Glen Research, and are referred to as linker arms. The oligonucleotides to which the cyanine dyes are linked are single stranded and generally contain between 8 and 50 bases. The oligonucleotides may be composed of ribonucleotides, deoxyribonucleotides, ribonucleotide derivatives, deoxyribonucleotide derivatives, or combinations thereof. Such oligonucleotides are well known in the art and can be prepared with commercially available nucleic acid synthesizers such as the 380B DNA synthesizer which is commercially available from Applied Biosystems of Foster City, Calif. In order to prepare cyanine dye-oligonucleotide conjugates of the present invention, the oligonucleotide is reacted with an appropriate linker or tether as a phosphoramidite reagent such that the linker covalently attaches to the oligonucleotide at its 5' end. Similarly, the linker can be covalently attached to an oligonucleotide at its 3' end or internally by tethering directly to a pyrimidine or purine ring using methods known by those in the art. A related method for internal labeling using isothiocyanate derivatives is described in a co-pending United States Patent Application filed on the same date herewith and assigned Ser. No. 08/276,271 and as described by Goodchild, J. (1990) Bioconjugate Chem. 1, 165-187. When a protected amine linker arm is used (attached at the 5' or 3' end, or through a purine or pyrimidine), the resultant product is then deprotected with an appropriate base such as ammonium hydroxide to leave a primary amine at the end of the linker. This resultant molecule is reacted with the cyanine dye under basic conditions and then purified by being passed through an appropriate column for example to remove unreactive dye and unlabeled oligonucleotide. Using fluorescence intensity measurements, life time fluorescence changes, or anisotropy, measurable differences can be detected between the single stranded cyanine dye-oligonucleotide conjugate and the product when this conjugate hybridizes to a nucleic acid target or binds to a protein or small molecular target. Generally a two-fold or greater fluorescence intensity increase is observed after hybridization of a single stranded oligonucleotide-cyanine dye conjugate to a complimentary unlabeled oligonucleotide. Fluorescence lifetime changes may also be observed and can be determined using dynamic fluorescence techniques. Significant changes in fluorescence polarization and anisotropy upon binding of the single stranded oligonucleotide-cyanine dye conjugates to oligonucleotides and other target molecules may also be used as means to detect the presence of these analytes. In addition to these qualitative differences between single stranded conjugate and double stranded product or bound target, quantitative values may also be obtained. One particularly useful form of fluorescence assay is the utilization of fluorescence polarization. Fluorescence polarization occurs when a fluorescent molecule is excited with polarized light which causes the emitted light from the fluorescent molecule to also be polarized. A quantitative determination of the polarization of the excited molecule can be determined by measuring the relative intensity of the emitted light parallel to and perpendicular to the plane of polarized light. An advantage of this type of assay is that it is homogeneous, that it does not require any separation steps. In such a polarization assay, polarizers are placed in the excitation beam and the emitted beam is measured through two polarizers; one parallel to the excitation polarizer and one perpendicular to the excitation polarizer. Polarization will be maximized if no molecular motion occurs and will be minimized if complete randomization occurs. These polarization assays measure rotational diffusion rates. Rotational diffusion rates relate to the size of the molecular species, that is smaller species rotate more rapidly than do larger species. Dynamic anisotropy and lifetime measurements are made by analyzing the decay of fluorescence intensity. These may be made either in the time domain (pulse method) or in the frequency domain (phase modulation method). Dynamic anisotropy measurements can be used to determine rotational correlation times. In general this value becomes larger as the rotational diffusion rate becomes slower. This increase can be correlated to binding of single stranded oligonucleotide-cyanine conjugates to target molecules. Polarization and anisotropy are also defined mathematically by the following equations: ##EQU1## where Ipa is parallel intensity and Ipe is perpendicular integrity. The relationship between anisotropy (r) and polarization (P) is also described by the equation: ##EQU2## The invention is further described by the following examples which are offered by way of illustration and are not intended to limit the invention in any manner. In these examples all percentages are by weight if for solids and by volume if for liquids or are used to refer to reaction yields, and all temperatures are in degrees Celsius unless otherwise noted. In this example oligodeoxynucleotides were prepared using an ABI380 B automated synthesizer (Applied Biosystems, Inc., Foster City, Calif.) using standard reagents supplied by the manufacturer, and purified by standard denaturing polyacrylamide gel electrophoresis techniques unless otherwise noted. The 5'-aminohexyl (C6) phosphoramidite reagent (ABI Aminolink 2�) was obtained from ABI. The 5'-aminopropyl (C3) linker phosphoramidite reagent was obtained from Glen Research (Sterling, Va.; product number 10-1903-90). NMR spectra for the compounds synthesized in the example were recorded on an IBM/Brucker WP-200SY (200 mHz) (Billerica, Mass.). High resolution fast atom bombardment (FAB) mass spectra (AIG, Inc., Raleigh, N.C.) were obtained with a high performance double focusing AMD 604 instrument with a resolution of 8000 amu. Low-resolution positive ion FAB mass spectra (FAB+) were obtained with a VG Trio-2 quadrupole instrument using either a glycerol or m-nitrobenzyl alcohol sample matrix. Preparative TLC was performed on glass-backed reverse phase PLKC18F silica gel plates (Whatman). UV/Vis spectra were obtained with a Hewlett Packard HP 8452A Spectrophotometer equipped with an HP 89090A cell controller for variable temperature experiments. 3-(1-(4-methyl-quinolinium))-propionic acid (1). Lepidine (2.95 gm, Aldrich) was mixed with 4.13 gm iodopropionic acid (Aldrich) neat. This mixture was heated at 80� C. for three hours under argon in an oil bath. The solid that formed was triturated with dicholoromethane and collected by filtration to give compound 1 as 5.2 gm of yellow solid (73%): 1 H NMR (DMSO-d6): ppm 3.01 (s,3H), 3.08 (t, 2H), 5.21 (t, 2H), 8.07 (m, 2H), 8.28 (t, 1H), 8.57 (dd, 2H), 9.43 (d, 1H), 12.5 (br s, 1H); 13 C NMR (DMSO-d6) ppm 19.8, 33.3, 52.8, 119.2, 122.4, 127.2, 128.9, 129.5, 135.2, 136.7, 149.3, 159.0, 171.4; LRMS (FAB+, glycerol)M+=216 m/z. (4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-1-quinolinium)-3-propionic acid (2). 1-(4-Methyl-quinoline)-propionic acid (1.0 g) and 1.0 g N-methyl-benzothiazolethiomethyl tosylate (Bader) were mixed together in 15 ml ethanol in a 50 mL round bottom flask. Triethylamine (0.1 mL) was added. Almost immediately the reaction mixture turned bright red. The reaction mixture was heated at reflux for two hours and cooled to room temperature. A red solid was isolated from the resulting (and foul smelling) solution. The yield of this material was 900 mg (47%) and only showed one spot near the origin on thin layer chromatography (silica gel, 9:1 dichloromethane/methanol). NMR (CD3 OD) 1 H ppm: 1.31 (t, 2H), 2.86 (t, 2H), 3.20 (t, 2H), 3.31 (s, 2H), 3.90 (s, 3H), 4.76 (t, 1H), 6.74 (s, 1H), 7.30, (m, 2H), 7.73, (m, 7H), 8.47 (dd, 2H); 13 C NMR (CD3 OD) ppm: 8.9, 20.0, 33.7, 38.0, 51.0, 88.8, 109.2, 113.3, 118.6, 125.5, 126.3, 126.7, 127.7, 129.0, 129.4, 134.1, 141.6, 145.4, 180.7, 189.8, 194.0; LRMS (FAB+, glycerol) M+=363 m/z (C21 H19 N2 O2 S). (4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-1-quinolinium)-3-propionic N-hydroxysuccinimide ester (3). Compound 2 (100 mg) and 125 mg 1,3-dicyclohexylcarbodiimide (DCC, Fluka) were added to a dry mixture of dichloromethane, tetrahydrofuran, and N, N dimethyl formamide and allowed to stir one hour at room temperature under argon. After one hour, 65 mg of N-hydroxy-succinimide was added and stirring continued overnight. The dark red solution was filtered leaving the desired NHS ester in solution. Solvents were removed under high vacuum conditions to yield a glossy solid. This solid was dissolved in dichloromethane and 2-propanol and stored in a refrigerator. Two crops of precipitated solid material were recovered for a total yield of 50 mg (�40%). Both fractions were analyzed by Low Resolution Mass Spectrometry (LRMS), Fast Atom Bombardment (FAB+) in glycerol. Both fractions showed M+ of 460, though the first fraction was more pure. The second fraction contained a higher molecular weight impurity suggested by a peak at 569 m/z. High resolution FAB+MS confirmed the identity of the molecular ion for the first fraction: 460.13296 m/z; calculated for C25 H22 N3 O4 S: 460.13276. TO-aminohexyl-5'-GTTCATCATCAGTAAC-3' (SEQ ID NO: 1) (4). The oligonucleotide was prepared using an ABI Aminolink 2� phosphoramidite reagent at the 5' end of the sequence. This oligonucleotide corresponds to nucleotides 1820-1835 of pBR322 as published in Watson, N., Gene 70, 398 (1988) and NCBI--GenBank Flat File Release 74.0, a typical small DNA plasmid, and is representative of typical target sequences. The crude product was separated from the column by treatment with ammonium hydroxide for 8 hours at 55� C. After passing the resulting mixture through an 0.45 micron filter and evaporation of solvent, a crude oligonucleotide was obtained by ethanol precipitation. Approximately 0.5 umol of the oligonucleotide was dissolved in 100 uL sodium carbonate buffer at pH 9.0 in an Epperndorf tube. A 0.5 mg aliquot of TO-NHS (Compound 3) was dissolved in 30 uL DMSO, added to the tube, and the mixture was left at room temperature in the dark for 2 hours. The mixture was passed through a NAP-5 Sephadex column (Pharmacia LKB Biotechnology) and eluted with 10 mM TAE. The first 1.0 mL fraction was concentrated and purified by polyacrylamide gel electrophoresis. TO-aminopropyl-5'-GGAATTCAGTTATCCACCATACGGATAG-3' (SEQ ID NO: 2) (5). The oligonucleotide was linked to thiazole orange with a 3-carbon linker arm obtained as a protected phosphoramidite reagent from Glen Research. Positions 9-28 of this oligonucleotide correspond to a Mycobacterium tuberculosis IS6110 target sequence represented by nucleotides 993-1012 of the sequence published in Thierry, D., Nuc. Acids Res. 18, 188 (1990). Subsequent deprotection was accomplished by reaction of the completed oligo from its column material by concentrated ammonium hydroxide at 55� C. for six hours. Deprotection cleaves the oligonucleotide from the solid support and removes the trifluoroacetyl protecting group from the aminoalkyl linker's nitrogen. Following speed vacuum concentration and ethanol precipitation, the reactive primary amine on the oligonucleotide was ready for reaction with the TO-NHS. A solution of this reactive dye 5.9 mg/150 μl DMSO (d6) (85.5 mM) was prepared. A 50 μl aliquot of the oligonucleotide (0.25 μM) in H2 O was diluted with 50 μl of 250 mM sodium carbonate buffer at pH 9.0. To this 0.125 μM oligonucleotide solution was added 10 μl of the dye solution. After vortexing the Eppendorf tube, it was covered in aluminum foil and allowed to sit at room temperature for 15 hours. The crude product was purified by the same procedure as the preceding Example. Similar thiazole yellow (TY)-oligonucleotide conjugates can be prepared by following the same procedures set forth above, but using N-methyl-benzoxazole-thiomethyl tosylate instead of N-methyl-benzothiazole-thiomethyl tosylate in the second step to produce compound 2. Compound 2 is then (4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene]-1-quinolinium)-3-propionic acid and compound 3 is (4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene]-1-quinolinium)-3-propionic acid N-hydroxy-succinimide ester. These experiments were performed on an SLM-Aminco model 8100 research grade spectrofluorometer with excitation at 510 nm. The fluorescence emission intensity was recorded from 515 to 600 nm and the fluorescence polarization was determined at 530 nm. The buffer for all measurements was 4 mM tris acetate, 0.1 mM EDTA, 50 mM NaCl at a pH of 7.8, and all measurements were at ambient temperature. The concentration of compound 5 (Example 1) and its complementary sequence were both 10 nM, with a sample size of 3 mL. Each value given for the fluorescence polarization is the average of three separate determinations. Under these conditions the unhybridized probe (compound 5) showed a steady state fluorescence polarization of 320.7 mP (milli-polarization units). The complementary sequence to compound 5 was added and the mixture was incubated in the dark for 30 min. At this time the fluorescence polarization was recorded again and had increased to 357.0 mP. The fluorescence intensity was also recorded for both the unhybridized and hybridized solutions (see FIG. 1). At 530 nm the change in fluorescence intensity was approximately a 4-fold increase. This experiment demonstrates that hybridization of a thiazole-orange oligonucleotide conjugate may be easily detected by changes in fluorescence polarization, fluorescence intensity, or both. Compound 5 from Example 1 (a 28-mer conjugated to thiazole orange) was tested using time resolved fluorescence techniques. Specifically, dynamic anisotropy was determined using frequency domain instrumentation. This instrumentation measures the local molecular environment near the fluorophore (thiazole orange) determining different rotational correlation times resulting from larger/smaller molecules. The dynamic anisotropy decays are measured and then interpreted based on experimental fitting curves (in this case Global Analysis) applied to these observed decays. The following Table 1 shows the results of this dynamic anisotropy testing. TABLE I______________________________________TO-28 mer T TO-28 mer/ComplementSingle Strand Double Strand______________________________________.O slashed. 3.4 ns (100%) .O slashed.1 0.5 ns (35%) .O slashed.2 14.8 ns (65%)______________________________________ The results of this experiment show that progressively as one goes from (1) single-stranded cyanine dye-oligonucleotide conjugate to (2) double-stranded product from the hybridization of conjugate to nucleic acid sequence target, significant changes of the rotational correlation times occur that indicate the formation of larger, more structured molecules. The invention disclosed herein is not limited in scope to the embodiments disclosed herein. Appropriate modifications, adaptations and expedients for applying the teachings herein in individual cases can be employed and understood by those skilled in the art, within the scope of the invention as claimed herebelow. __________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 2(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GTTCATCATCAGTAAC16(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 28 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GGAATTCAGTTATCCACCATACGGATAG28__________________________________________________________________________ Patent Citations
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07/27/1999STATEMENT BY CAROL M. BROWNER U.S. EPA ADMINISTRATOR ON FINDINGS BY EPA'S BLUE RIBBON MTBE PANEL July 27, 1999 On November 30, 1998, I appointed a Blue Ribbon Panel of leading experts to investigate concerns raised by the discovery of MTBE, a gasoline additive, in some water supplies. I want to congratulate and thank each member of the Panel for their excellent work, beginning with the Chairman, Dr. Daniel Greenbaum of the Health Effects Institute of Cambridge, MA. The Panel recognized the significant benefits of reduced auto emissions and improved air quality from cleaner burning gasoline and the role oxygenates, like MTBE and ethanol have played. But the Panel also found that when gasoline leaks and spills from sources such as underground storage tanks and gas cans, MTBE can, in fact, pose risks to water supplies. I stated when the Panel was assembled that my goal was to protect public health and the environment by ensuring that Americans have both cleaner air and cleaner water – and never one at the expense of the other. And that is what EPA intends to do. The recommendations I received from the Panel confirm EPA's belief that we must begin to significantly reduce the use of MTBE in gasoline as quickly as possible without sacrificing the gains we've made in achieving cleaner air. EPA is committed to working with Congress to provide a targeted legislative solution that maintains our air quality gains and allows for the reduction of MTBE, while preserving the important role of renewable fuels like ethanol. In addition, EPA will improve gasoline leak protection and remediation programs and provide the states with maximum flexibility under current law that will make it easier to voluntarily reduce MTBE and use cleaner gasolines with other additives. These actions will ensure that millions of Americans will continue to breathe healthier air in their communities while also receiving improved protection of their water supplies. R-85 ### |
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Joe Colborne
Flames say line brawl vs. Canucks turned season around, was 'good for hockey'
Apr 14, 2014, 1:06 PM EST
This should…generate some comments.
Carlyle to Colborne: Get on the bus…because the bus is leaving…and you want to be on that bus…it's a good bus
Sep 19, 2013, 4:19 PM EST
Or something like that.
PHT Morning Skate: Where a Leafs prospect wound up being injured pretty badly
Sep 18, 2012, 9:00 AM EST
How do you feel about some puck with your Thanksgiving turkey?
PHT Morning Skate: Panthers are so hot, they're stripping in practice
Nov 23, 2011, 7:00 AM EST
Comments OffThe Panthers must be hot because they're busy stripping down during shootouts in practice


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Beauty Bridge.co m
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Read Review of Beauty Bridge.co m
Beauty Bridge
Reveal Your Natural Beauty
Dec 16, 2006
Ease of Ordering:
Customer Service:
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Full Review
It's no secret that I'm highly attached to beauty products. When a manufacturer releases new stuff, I'm
all over it
. Time-permitting, I like to spend bazillions of hours physically picking up and putting down all of these
new products. Most of the time though, I don't do any serious beauty product purchasing unless it is online - and specifically from my two favorite stores -
Because these two Online Stores pretty much carry the world between them, I don't have the need to stray towards others.
One day, while rating some online surveys on
, I came across this merchant -
Beauty Bridge.com
. Curiosity killed my cat and I felt the need to explore.
About Beauty Bridge
The newly revamped website doesn't tell us much about
Beauty Bridge
the company. What I did find out is that it's been around since 2003 and is headquartered in Nutley, New Jersey.
Beauty Bridge
is highly focused on specific products and product lines.
Reveal Your Natural Beauty
is their slant on the world and you'll find that the product lines they offer very much match their philosophy.
You can purchase products for skin care, make-up, mineral cosmetics, bath & body, and hair & nail. Featured brands include: Bare Escentuals, Bioelements, Dirty Girl by Blue Q, GloMinerals, Primal Elements, ZenMomma, Baby Cakes, and Illuminare. There are
others - just wanted to give you a hint of the variation.
Experiencing the Online-ness Of It All
Web Aesthetics
Beauty Bridge
recently had their own face-lift of sorts. Today's site is pleasantly laid out, with soft and subtle colors, and easy to read details about the products that they offer up for retail therapy. Navigating in and out of product lines was easy - as was adding items to my cart and checking out.
Customer Service
Customer Service comes in various forms. If you're looking for information on your order, or have a question about a product, you have the opportunity to chat live with someone. The only downfall is if you're shopping in the wee hours and suddenly, you have a burning question that must be answered now! In those circumstances, you can email them and they'll get back to you pretty quickly.
Products - Availability
I mentioned a few of the product lines that they offer. Overall, they tend to be the higher-end things that you won't find in many places and certainly, not in big, bulky amounts. I had no issue with product availability (for my own order) however, unlike other sites, they don't tend to let you know up front whether or not the product is in stock (or not).
Pricing, Shipping, Options for Payment, Returns, Promotions
With regards to the pricing of their products, they tend to run the same as other places that stock them. I mean, you're not going to find
for $1.00 anywhere ;).
You have various options for shipping and you pay accordingly but aside from knowing your options,
Beauty Bridge
did a good job (in my case) of communicating shipping expectations around the holidays and PRIOR to placing the order.
Beauty Bridge
accepts Mastercard, Visa, Discover, American Express, Discover, Money Order/Check, and Paypal.
Returns are a
very tricky
adventure. I never had to return my order, but if you find yourself in this boat, make sure that you
read their requirements. In some cases, the manufacturer of the products don't allow return of the product - in other cases, you can return items because they didn't meet your expectations. Again, I highly recommend you read this section.
Occasionally, they have promotions going on. They're not nearly as big and splashy as what you might find elsewhere however they come in handy and are appreciated.
During my first visit, I received free shipping with purchases over $75 and 5% off my first order.
Other Information
You can peruse their Blog for great (and may I say - very applicable) beauty advice, or, just recently started - a bulletin board forum where you can interact with other consumers who are all beauty product obsessed.
The End
My very small box arrived on-time, carefully packaged, and in pristine condition. I was satisfied with my experience from start to finish and accordingly, have recommended this e-tailer as an option to those who are just as beauty-obsessed as I am.
What product did you purchase or try to purchase?
Bioelements, Too Faced
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Sen. Dodd: Troop Increase Continues 'Failed Policy' January 31, 2007 Sen. Christopher Dodd (D-CT) says President Bush's plan to increase troops in Iraq is a "continuation of a failed policy." Dodd, a candidate for the Democratic presidential nomination in 2008, discusses the Iraq war and his presidential aspirations. Listen Loading… Download Playlist
Iraq Stuart Bowen on Problems in Iraq January 31, 2007 Stuart Bowen, Special Inspector General for Iraq Reconstruction, has just issued his quarterly report investigating waste, fraud and security problems in the reconstruction efforts. Listen Loading… Download Playlist
Hurricane Katrina & Recovery Efforts Branford Marsalis' Changing New Orleans January 31, 2007 Jazz musician Branford Marsalis discusses his commitment to musicians hit hard by Hurricane Katrina and helping rebuild his home town of New Orleans. Listen Loading… Download Playlist
Books Author on Growl Power of 'Cheetah Girls' January 31, 2007 Author Deborah Gregory talks about the inspiration behind her young-adult fiction series, The Cheetah Girls. Listen Loading… Download Playlist
Bill Gates on Business and Charity January 30, 2007 Microsoft chairman Bill Gates joins Alex Chadwick to talk about Windows Vista, the company's new operating system. Gates also talks about his reaction to the Apple-Microsoft ads and the recent controversy surrounding the Bill and Melinda Gates Foundation and its investment policies. Listen Loading… Download Playlist
Race The Harlem Renaissance, On and Off the Court January 30, 2007 Not just a literary movement, the Harlem Renaissance was also the name of a famous ballroom in the New York City neighborhood and a barrier-breaking basketball team. Kareem Abdul-Jabbar has written a book that chronicles their histories. Listen Loading… Download Playlist
Politics Senator Chuck Schumer January 30, 2007 Sen. Chuck Schumer (D-NY) was elected to Congress at the age of 29 and served in the House of Representatives for eight years. He just began his second term as a Senator. He is a member of the Senate Democratic Leadership team, and sits on the Senate Finance Committee; the Committee on Banking, Housing, and Urban Affairs; and the Judiciary Committee. His new book is Positively American: Winning Back the Middle-Class Majority One Family At a Time. Listen Loading… Download Playlist
Singer Elisabeth Withers Goes Solo January 30, 2007 Farai Chideya talks with Elisabeth Withers, the Tony-nominated actor whose debut album is titled It Can Happen to Anyone. Withers played Shug Avery in the Broadway version of The Color Purple. Listen Loading… Download Playlist
'Loose Nukes' Threat Growing, Nunn Says January 30, 2007 A Russian is held in the former Soviet republic of Georgia for offering to sell weapons-grade uranium. The incident highlights the problem of securing nuclear material, says former Sen. Sam Nunn. Listen Loading… Download Playlist
Politics President Bush, Part 2: Domestic Issues January 29, 2007 President Bush sits down with NPR's Juan Williams for his first broadcast interview since the State of the Union. They discuss the environment, health care, the federal budget and the quality of intelligence coming out of Iran. Listen Loading… Download Playlist
Full Transcript: NPR Interview with President Bush January 29, 2007 The following is a full transcript of an interview with President George W. Bush, conducted by Juan Williams on Monday, Jan. 29, 2007.
Arts & Life Author Thomas Ricks and Singer Josh Ritter January 29, 2007 Washington Post reporter Thomas Ricks discusses his book, Fiasco: The American Military Adventure in Iraq, and he is joined by singer-songwriter Josh Ritter who performs "Girl in the War." Listen Loading… Download Playlist
Iraq Ambassador Khalilzad Discusses Iraq January 29, 2007 Zalmay Khalilzad has served as U.S. ambassador to Iraq since 2005, and is set to leave that post to become the U.S. ambassador to the United Nations. Khalilzad talks about how the security and the political situation in Iraq has evolved during his tenure. Listen Loading… Download Playlist
Juan Williams on Bush Interview January 29, 2007 NPR correspondent Juan Williams talks about his conversation with President Bush, the president's first broadcast interview since last Tuesday's State of the Union Address. Listen Loading… Download Playlist
Justine Simmons of 'Run's House' Pens Book for Kids January 29, 2007 Justine Simmons is probably best known from the MTV reality series Run's House. The show follows the family life of Simmons and her husband, Reverend Run, a member of the groundbreaking rap group Run DMC. Simmons talks about her new, illustrated children's book, God, Can You Hear Me? Listen Loading… Download Playlist


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Get free worksheets plus activities, articles and science projects.
Bang on your flower pots, string your your shoebox guitar, and make some bottle maracas with this week's round-up of music-making activities and printables! Remember that some great learning can come from making some great music and that's what this week is all about.


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Mark _ June 07, 2013 4:22 PM It wouldn't be the first time that a team gets punished after following Charlie's advice. Reply Report abuse 12 agree 1 disagree
richard June 07, 2013 4:40 PM Charlie is not the fia. surely only the fia would have been authorised to overlook the rules? Reply Report abuse 5 agree 9 disagree
Fernandabulous June 07, 2013 5:26 PM he authorised things for ross brawn before while giving others a different interpretation in his role as an fia representarive Reply Report abuse 4 agree 0 disagree
AbdominalSnowman June 07, 2013 5:50 PM I told you guys they would have got the OK from somebody in a moment of casual nonsense talking. Here it is. Reply Report abuse 0 agree 1 disagree
107SS2009 June 07, 2013 7:20 PM Charlie Whiting is not the FIA but he is sure the man responsible for such testing matters in fact he is the one to contact and ok or not such matters.It could very well turn out that Mercedes has pulled a fast one for next year and RBR and FERRARI are not happy. This year Mercedes is better, but still away off challenging for the titles, the chance to have data for next year is too good an opportunity to pass up. Reply Report abuse 3 agree 3 disagree
107SS2009 June 07, 2013 7:23 PM And if Bernie calculates that the audience is up on last year than he will say there is no problem to fix! Reply Report abuse 3 agree 0 disagree
over2u June 09, 2013 12:37 AM Charlie works for Mr.F1 takes all instructions from the ring master, the target appears to be Ross B, this saga may be a reason for Lauda and Wolff to get rid off RB. Reply Report abuse 0 agree 0 disagree


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By Raqiyyah R. Pippins, Kelley Drye & Warren LLP
Published February 23, 2012
Attorney Raqiyyah R. Pippins of Kelley Drye & Warren LLP says that when it comes to enforcement actions, it's all in the eyes of the beholder. Read More »
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Published February 20, 2012
Nick Morante explains how powders can also boost the perceived efficacy of products. Read More »
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Published February 9, 2012
Looking to the farmer's market as the new pharmacopeia. Read More »
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For Oksana Panasenko, founder of Dairyface skin care products, it's not enough to use fresh, natural ingredients' products must be refrigerated too! Read More »


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Life on Titan? This Moon May Hide a Strange Form of Life
Wearable Tech You'll Actually Want to Wear
State-sponsored computer spies, hacker collectives, criminals, and random trolls all mine the Internet for personal data. Some hackers trick users into giving up personal info; others steal it from government and business databases.
Searches and online email accounts provide rich sources of data for advertisers; weak password protection can also make personal online accounts prime targets for hackers.


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Reshuffle at top as ethical fund slides
PUBLISHED: 26 Apr 2012 00:04:01 | UPDATED: 26 Apr 2012 04:14:46PUBLISHED: 26 Apr 2012 PRINT EDITION: 26 Apr 2012Australia's largest ethical fund manager, Hunter Hall International, has made changes to its management and investment teams after a period or poor returns and falling profits.
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Title | Date | Body | Location |
Bluegrass Under the Stars Concert and Fireworks | Apr 4 2015 - 6:00pm - 10:00pm |
Saturday, April 4, 2015 Free Admission Featured Bluegrass Bands This year's lineup and band website links (in order of appearance) Special Headliner Presentation by Country Music Television Also featuring a pre-concert performance by the Sweet Potato Pie Kids Other Concert Information: Concert Ends with a Fireworks Extravaganza! Free Admission PARKING LOT CONCERT: Bring a chair or a blanket Concessions Rain or Shine (rain location: Student Center) No alcohol, coolers, or pets allowed No Solicitation Tobacco Free Campus For more information, visit www.tctc.edu/bluegrass This concert is offered in conjunction with the Historic Pendleton Spring Jubilee. Price: Free |
Tri-County Technical College |
Passport to the Arts | Mar 6 2015 - 6:00pm - 9:30pm |
It is time for the most exciting art event of the year, Passport to the Arts! The annual Passport to the Arts continues to be an exciting and popular "Town and Gown" event. Join The Center for Visual Arts – Lee Gallery and The Arts Center of Clemson for an evening of fine art, entertainers, live music, drinks, and exceptional food showcased at four different locations. Grab your passport and let the CAT (Clemson Area Transit) Bus take you on a visual arts tour of Clemson for a fun filled evening. All buses feature entertainment making your ride to each venue even more enjoyable. Locations this year include The Arts Center, CAT Bus, Lee Gallery and OLLI in Patrick Square. SORRY - THIS EVENT IS COMPLETELY SOLD OUT! |
Various Clemson Locations |
Board of Zoning Appeals Meeting - March 19, 2015 | Mar 19 2015 - 6:00pm - 7:30pm |
City Hall - City Council Chamber |
City Council Meeting | Apr 6 2015 - 7:30pm - 9:30pm |
AGENDA not posted. |
Council Chambers, City Hall 1250 Tiger Boulevard |
City Council Meeting | Apr 20 2015 - 7:30pm - 9:30pm |
AGENDA not posted. |
Council Chambers, City Hall 1250 Tiger Boulevard |
Easter Egg Hunt | Apr 4 2015 - 10:00am - 12:00pm |
Click here for more information. |
Nettles Park, 102 Nettles Park Road |


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Auction of 'Breaking Bad' props to benefit New Mexico Goodwill stores
Published 09/13/2013, Associated Press
Memorabilia from the AMC hit television series "Breaking Bad" is scheduled to be auctioned to benefit Albuquerque-area Goodwill stores after the nonprofit helped with production.
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Wednesday, January 25, 2012
After watching last night, I have come to a simple conclusion. WE ARE SCREWED! There are no two parties, just one big blob trying to eat us, no matter which way we turn. Apparently, no one in this country has really read the history of Germany in the 30's. We are reliving it. These politicians are nothing but common criminals. Enough is enough. I am spitting myself out of the system. Whatever happens in the political realm of this fantasy land will not affect how I continue to raise my family as Independent Thinkers and live by the Constitution in Freedom and Liberty.
Thursday, September 8, 2011
Is there anyone running for President that actually wants to unselfishly serve our Counrty and protect our Constitution or do they just want the title?
Monday, October 4, 2010
Communism's 20%
This video gets us used to 20% of our population being killed to cowl the other 80%. Over 60 million people died in China. How many do the Communist's want to kill in America?
Friday, April 30, 2010
As I check the voting record on the Puerto Rico bill,
I ask myself which Congressmen are voting solely for the up-coming elections. I see a pattern of political fence sitting, designed to show plausible deniability. Any Congressional member "Not Voting" has shown their true colors. They are pure politicians without a shred of character. We are in the midst of finding out that "Character DOES Matter" with the people we have been electing over and over, some have over 40 years in office. It is not about how much funding or how interesting the visuals are in their commercials, it is about personal principles, values, and morals. I haven't seen a lily-white, cleanly principled "politician", yet. I don't think such a person exists. We need to look at people standing up for the first time in their respective careers and are willing to represent their communities out of disgust over the systems failures. Corruption doesn't trickle down from the top, it starts at the bottom and works it's way up the ladder. A School Board member that has shown a preference for special interests and unions isn't going to change if you elect them to City Council. A City Council member or County Commissioner with questionable ties to lobbyists isn't going to stop enjoying the perks if they get into Congress. Politics should not be a career, it should be an Honor and a Duty and a BURDEN to the soul.
Power attracts the corruptible. Suspect all who seek it. We should grant power over our affairs only to those who are reluctant to hold it and then only under conditions that increase the reluctance.
Frank Herbert 1985
Wise words to consider in our Country's present situation of corruption and moral degradation.
Wednesday, April 21, 2010
Where is our decency?
Why are we reading about these stranded travelers going broke? Where is our decency and sense of charity? How much effort would it take for New Yorker's to give a stranded traveler a couch to sleep on and a meal? Why are our clergy and congregations not reaching out to shelter these people? How can we just leave people to live in an airport? No one likes to have a long layover, let alone have to sleep on the floor with your luggage for days. I am saddened to see how far we have moved from the foundations of our country. We have always been known for our charitable nature. We have always been the first to send help in a crisis no matter where in the world it is needed, yet here we sit waiting for the government to set up some kind of "tent city" for these people who came to our land to vacation, visit family, do business, etc. I am ashamed by the inaction of my fellow countrymen.
Wednesday, November 11, 2009
Happy Veteran's Day
Thank You
Tuesday, November 10, 2009
Happy Birthday USMC
MARINE CORPS ORDER No. 47 (Series 1921)
HEADQUARTERS U.S. MARINE CORPS Washington, November 1, 1921
The following will be read to the command on the 10th of November, 1921, and hereafter on the 10th of November of every year. Should the order not be received by the 10th of November, 1921, it will be read upon receipt.
On November 10, 1775, a Corps of Marines was created by a resolution of Continental Congress. Since that date many thousand men have borne the name "Marine". In memory of them it is fitting that we who are Marines should commemorate the birthday of our corps by calling to mind the glories of its long and illustrious history. The record of our corps is one which will bear comparison with that of the most famous military organizations in the world's history. During 90 of the 146 years of its existence the Marine Corps has been in action against the Nation's foes. From the Battle of Trenton to the Argonne, Marines have won foremost honors in war, and in the long eras of tranquility at home, generation after generation of Marines have grown gray in war in both hemispheres and in every corner of the seven seas, that our country and its citizens might enjoy peace and security. In every battle and skirmish since the birth of our corps, Marines have acquitted themselves with the greatest distinction, winning new honors on each occasion until the term "Marine" has come to signify all that is highest in military efficiency and soldierly virtue. This high name of distinction and soldierly repute we who are Marines today have received from those who preceded us in the corps. With it we have also received from them the eternal spirit which has animated our corps from generation to generation and has been the distinguishing mark of the Marines in every age. So long as that spirit continues to flourish Marines will be found equal to every emergency in the future as they have been in the past, and the men of our Nation will regard us as worthy successors to the long line of illustrious men who have served as "Soldiers of the Sea" since the founding of the Corps.
JOHN A. LEJEUNE, Major General Commandant
Healthcare In America
Monday, October 19, 2009
Belated Columbus Day
As I reel from the White House's attempts at overwhelming the system,daily, I forgot to wish everyone a Happy Columbus Day! I searched high and low to find the most appropriate picture to show how excited our "citizens" are to be here celebrating the momentous day!
Wednesday, October 14, 2009
An Open Letter to Maine
Dear Residents of Maine,
I am extremely curious on the philosophy of your Democratic Party. Do you even have one? They must be so radically liberal you don't let them out of the state. Really, who needs Democrats when you have Susan Collins and Olympia Snowe? I thought we had it bad here in Ohio, but I see the grass is actually greener on this side of the fence, for the time being.
Yours Truly,
Ohio Conservative
Barely Represented
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Lyrics Depot | |
Lyrics » C Murder Lyrics » Interlude Lyrics |
Lyrics Depot is your source of lyrics to Interlude by C Murder. Please check back for more C Murder lyrics. |
Interlude LyricsArtist: C MurderAnd let me put my dick in your mouth Please don't you act like you shy And let me bust a nut in your eye (2x's)
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Project Information
UH Hilo Home > Faculty and Staff > Facilities Planning & Construction
Kapiolani & Kawili Street Sidewalk & Crosswalk Improvements
This project will provide an ADA compliant sidewalk along Kapiolani Street and make improvements to the Kapiolani/Kawili Street intersection.
Project Code: - UHH-2014-404
Approximate Cost:
Funds | Approximate Cost |
Planning/Design | $150,000.00 |
Construction/Equipment | $1,600,000.00 |
Total: | $1,750,000.00 |
Consultant(s): Okahara & Associates, Inc.
Project Manager: Michael Murakami
Construction Manager: to be determined
Contractor(s): to be determined
Affected Area(s): to be determined View Map
Estimated Construction Time: September 2015 to May 2016
Last Updated: January 27th, 2015 4:08pm
Note: The information above is subject to change. Please contact the Facilities Planning & Construction Office for current information.


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The classification
Abstract | References | Similar Articles | Additional Information Abstract: Cuntz-Krieger algebras with exactly one nontrivial closed ideal are classified up to stable isomorphism by the Cuntz invariant. The proof relies on Rørdam's classification of simple Cuntz-Krieger algebras up to stable isomorphism and the author's classification of two-component reducible topological Markov chains up to flow equivalence.
Retrieve articles in Proceedings of the American Mathematical Society with MSC (1991): 46L35, 54H20, 46L55 Retrieve articles in all journals with MSC (1991): 46L35, 54H20, 46L55
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Coffee Delights Appliqued Women's Cotton Bathrobe
Owl Printed Plush Micro-Terry Girl's Bathrobe
Woman's Fifi in Paris Short Bathrobe
Women's Lightweight Sweatshirt Knit Bathrobe in Heather Gray
Monkey on Cloud Printed Women's Shower Wrap
Jumbo Dots Print Hooded Robe in Navy & Fuchsia
Pastel Dot Printed Women's Soft Minky Bathrobe
Women's Cat Appliqued Terry Cloth Bathrobe
Football Appliqued Plush Boy's Bathrobe
Unisex Solid Cotton Terry Loop Heavy Long Bathrobe
Marled Knit Henley Top and Pant Lounge PJ Set in Charcoal
Women's Lovely Script Applique Bathrobe
Unisex Terry Velour Hotel Spa Bathrobe
Boy's Sports Bathrobe
Hotel Spa Women's Bathrobe with Terry Loop Lining
Women's Sweetheart Appliqued Cotton Terry Short Bathrobe
Women's Bath Duck Appliqued Bathrobe
Unisex Zero Twist Terry Hooded Bathrobe
Women's Kimono Style Knit Houndstooth Lightweight Short Bathrobe
Women's Puppy Applique Cotton Terry Long Bathrobe
Crazy Hearts Jacquard Plush Microterry Women's Short Bathrobe
Solid Zero Twist Terry Loop Women's Shower Wrap with Gingham Trim
Solid Terry Loop Women's Plus Size Shower Wrap with Bow
Boy's Plaid Print Bathrobe


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South Nassau Communities Hospital
Tri-State Hospitals Researching Expansion Of Age Range For CT Scan Insurance Coverage
Insurance companies are now paying for costly CT scans for longtime smokers over the age of 55.
NY Sisters Deliver Babies Hours Apart At Same LI Hospital
Michelle Clarke, 29, delivered daughter Savannah at about 1:30 p.m. Friday, just two hours after younger sister Christina Santos-Santiago delivered little Nicolas.
L.I. Hospital Says Thousands Of Patients May Be At Risk Of Blood Contamination
South Nassau Communities Hospital is sending out over 4,000 letters to patients who may have been exposed to dangerous viruses, including hepatitis and HIV, through insulin shots.
L.I. Hospital: Reused Insulin Pen Part May Mean Hepatitis, HIV Risk For Patients
The South Nassau Communities Hospital has advised that some patients who received injections from an insulin pen should get tested for hepatitis and HIV.
Long Beach Residents Demand Sandy-Damaged Medical Center Be Reopened
The barrier island of Long Beach is taking steps forward this summer in its Hurricane Sandy recovery. Churches, beaches, and the boardwalk have all slowly reopened. But not it's only hospital.