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South Africa's samango monkeys live on what ecologists call a "vertical axis of fear." They live and eat in trees, and if they climb too high looking for food, they're liable to get attacked and eaten by an eagle. If they venture too low, it's leopards and desert lynxes they have to worry about.... READ ON
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Nebraskan Julius Sterling Morton believed that planting trees could save America, and he convinced the state to celebrate Arbor Day in April 1872. By planting trees, Morton hoped to prevent erosion, preserve the topsoil, and break some of the raging winds across the plains. While Arbor Day wasn't officially adopted in the United States until 100 years later, Morton was onto something. A recent study finds that trees can save America, though not quite in the ways Morton envisioned. Instead, the study found t... READ ON
In the tropical forests of Central and South America, a fantastic looking beetle begins its life in the drabbest of places.... READ ON
When love is in the air for certain moth species, the female secretes a pheromone to signal that she's ready to make little moths. An interested male will approach and "sing" her a courtship song composed of a series of ultrasonic pulses. ... READ ON
After a 2011 vote, Toyota announced that the official plural of Prius was Prii.

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text:
- Rihanna
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|Song details
|Rihanna feat. Chris Brown and Jay-Z
|Good Girl Gone Bad [2007], Track 2
|Dance, Pop, R&B
|8,212 (−5,834) history »
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|Song lyrics
(Uh-Huh-Uh-Huh) Remix, Yeah
(Uh-Huh-Uh-Huh) Rihanna, Chris Brown
Good Girl Gone Bad (Uh-Huh-Uh-Huh)
Take three, Action (Uh-Huh-Uh-Huh), Ho
No clouds in my storms
Let it rain I hydroplane into fame
Coming down with Dow Jones (Eh-Eh-Eh)
When the clouds come we gone
We Rocafella, we fly high than weather
And G5's are better (Eh-Eh-Eh, Eh)
You know me (You know me)
An anticipation for precipitation
Stack chips for the rainy day
Jay, Rain Man is back (Eh-Eh-Eh)
With Little Ms. Sunshine
Rihanna where you at? (Eh-Eh-Eh, Eh)
You have my heart
And we'll never be worlds apart
Maybe in magazines
But you'll still be my star
Baby 'cause in the Dark
You can see shiny Cars
And that's when you need me there
With you I'll always share, because
When there's sunshine we'll shine Together
Told you I'll be here forever
Said I'll always be a friend
Took an oath I'mma stick it out 'till the end
Now that it's raining more than ever
Know that we'll still have each other
You can stand under my umbrella
You can stand under my umbrella
Under my umbrella
Under my umbrella
Under my umbrella
You're becoming a dream to me
A fairytale fantasy
Nothing can ever compare
An image to my memory
Girl I'm asking could you be my queen?
A vision on a magazine
That's when I'll be there
It's something we both share, because
When there's sunshine we'll shine Together
You know I'll be here forever
Although it's a lot of rain outside
Girl it's getting late you can stay the night
But you can dip out anytime whenever
I can call a car I ain't tryna stress ya
I'm looking for the one with the glass slipper
Baby girl you can be my Cinderella
You can be my Cinderella
You can be my Cinderella
You can be my Cinderella
You can run into my Arms
It's okay don't be alarmed come into Me
There's no distance in between, our love
So go and say my name some
More I'll be all you need and
Mo-o-o-o-o-o-ore (Ah, you ready?), because
When there's sunshine we'll shine Together
Told you I'll be here forever
Said I'll always be a friend
Took an oath I'mma stick it out 'till the end
But you can dip out anytime whenever
I can call a car I ain't tryna stress ya
I'm looking for the one with the glass slipper
Baby girl you can be my Cinderella
Under my umbrella
You can be my Cinderella
Under my umbrella
It's raining-raining (Raining)
Ooh Baby it's raining-raining (Raining)
Baby come into Me (Oh)
Come into Me (Na-Na)
It's raining-raining (Na-Na)
Oh Baby it's raining-raining (Na-Na)
You can always come into Me
Come into Me
submitted on June 13th, 2007
corrected by Edward Louk on January 30th, 2015
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|18 Comments
|0 likes - like | 24-06-2009 18:10 risamarisa
331 posts
|so much for rihanna being his cinderella....he beat the fuck out of herrr!
|0 likes - like | 07-05-2008 01:30 yaia
56 posts
|love her vocals but its so lame with chris brown
besides i am so tired of this song!!!!!!!!
|0 likes - like | 20-04-2008 10:50 Arual_92
2,312 posts
|i prefer this version with chris brown.. bt i guess thats jus coz i luv his voice cz rihanna and jay-z version without chris brown rox anyway
|0 likes - like | 12-12-2007 23:47 itsLauren
62 posts
|lovedd itt.
&& soo did my momm =]
|0 likes - like | 08-09-2007 11:42 Betnii
28 posts
|song well gurd
|0 likes - like | 04-09-2007 00:46 KatherineHope
1 posts
|It bothers me when people say that Rihanna has "nothing to offer". The girl can SING and she's unlike anybody else out there. Whenever Rihanna comes on the radio, she has a very recognizable sound. She can incorporate a fun groove into almost any song she sings. There hasn't been one Rihanna song that I haven't fallen in love with. Needless to say, I love this girl and she will definately be one of the biggest popstars of the decade, and hopefully she will be around for much longer. She has so much talent and special quality. She just keeps on getting better with each album too, and I feel as though she has yet to hit her peak.
I have to say that I prefer the original version of Umbrella way more over this. I'm sorry but Chris can't pull off the "ella ella eh" part like Rihanna can, and he just sounds awkward through the whole song. Umbrella was never meant to be a song that showcased a singer's full vocal potential, but rather a fun song. Chris doesn't even sound like he's having fun with it. Rihanna can take any song and make it fun and catchy, that's what I love about her.
This song loses points from me because of Chris. He's definately a talented young artist, but he's no Rihanna.
And regardless of what "musical background" you claim to have, you can't deny that Rihanna is a bundle of talent. Just listen to her new CD
|0 likes - like | 04-09-2007 00:06 SnowCone
1 posts
|Chris Brown blew this jam up. Rihanna and Jay-Z need to send props. Watch out cause that bad young brotha ain't done yet....
|0 likes - like | 30-08-2007 03:55 Svg12
2 posts
|Thanks hopeincubus for posting a really valuable review, We have the same opinion.
|0 likes - like | 29-08-2007 17:51 hopeincubus
1 posts
|I think that this version of the song is about 4 times better than the original. I'm not a fan of rihanna's or chris brown but this song is pretty good. It very melodic and having at least one good singer (chris brown) always helps. I have a musical background so i feel confident when i say I do not believe rihanna has anything special to offer as far as singing goes.
Nonethelss THIS version of the song is good.
|0 likes - like | 28-08-2007 19:41 Svg12
2 posts
|Chris Brown didnt ruin anything, he is very cute and he is a great young singer. It looks that anybody in here knows about singing. He actually put some flavor to the original song, now its much better. And about Ne-yo I dont think so Chris was the perfect to the song
|0 likes - like | 13-08-2007 18:29 knot2keen
13 posts
|My favorite part of the song is when they sing together toward the end
but chris brown sounds kinda whiny, thats what kills it for me
|0 likes - like | 01-08-2007 18:21 sassybrunette
40 posts
|yeah, i love this one and the original verrryy much! i never get tired of them...but this one may be a little better
[[my opinion]] dont hate
|0 likes - like | 27-07-2007 16:17 kevina
183 posts
|poor chris.. he is a good singer but not this time. he ruins the song..
|0 likes - like | 12-07-2007 08:06 shinitaa
42 posts
|lovee thisss
|0 likes - like | 11-07-2007 20:52 DC40483
1 posts
|I like love this song i think it was very good!!! it's like mt # 1 song!!!
|0 likes - like | 02-07-2007 10:15 ley31
11 posts
|the song is actually ok..
|0 likes - like | 26-06-2007 03:42 DaMoZ
9 posts
|This is ok. Rihanna rox chris brown not so much. i think that they should have made this remix with Ne*yo then i think it would be better.
|0 likes - like | 25-06-2007 17:52 ANRON
240 posts
|Eww i hate chris brown in this song. he ruins it


text:
This invention involves the elution of macromolecules such as proteins, deoxyribonucleic acid(DNA), ribonucleic acid(RNA) from solid phase matrix such as polyacrylamide, agarose and membranes such as polyvinyl difluoride (PVDF) in an electric field. The elution chamber shown in FIG. 1 consists of two separate screws 9, 9a and attachable chamber 1. The screw well 8 holds the gel matrix containing the macromolecules and is separated by the bottom chamber 1 with a membrane 5 with a nominal molecular weight cutoff higher than the macromolecule. On the other hand, the macromolecule is retained in the well 1 by introducing another membrane 6 having a molecular weight cutoff lower than the molecular weight of the macromolecule. Alternatively, macromolecules separated on membrane, e.g. PVDF, are held in a Teflon or plastic membrane holder 11. This holder can fit inside well 8 in screw 9. In addition, chamber 1 contains a well 7. The macromolecules migrate through the electrophoretic field and are concentrated in well 7. Since the volume of the well 7 is predetermined (10 ul--one ml or more), results in concentration of the macromolecules. Next, the chamber 1 is unscrewed from the top screw 8 and closed with a screw without a hole 13, and the entire device is submerged into a dialysis buffer. Following dialysis for a given time, the fluid above the bottom screw 9a itself and the membrane beneath it are removed. The sample is taken out from hole 7. If further concentration of the sample is required, then following removal of the fluid above the screw 9a, the entire device is transferred to a vacuum desiccator and concentrated to any desired volume.
1. An electroelution device for the electroelution of macromolecules from a support, comprising a chamber and upper and lower releasably securable membrane retaining members, said chamber, and at least one of said upper and lower membrane retaining members having a through-hole for passage of a fluid, said chamber through-hole terminating at a membrane well at each of its ends and having a diameter less than that of said membrane wells, each of said membrane wells having a sidewall that is provided with a shoulder for receiving a membrane of a prescribed molecular weight porosity and a corresponding one of said membrane retaining members along a common axis such that the membrane is interposed between said shoulder and said membrane retaining member, said sidewalls and said membrane retaining members each having complementary-configured engagement surfaces for effecting engagement and retention of the respective membrane retaining members received therein.
2. The device of claim 1 wherein said membranes have molecular weight exclusions such that said macromolecules are retained within the through-hole of said chamber after electroelution.
3. The device of claim 2 wherein said membranes have different molecular weight exclusions.
4. The device of claim 1 which further comprises a holder for holding said support.
5. An electrophoresis tank containing the electroelution device of claim 1.
6. The electroelution device of claim 1 wherein the through-hole of at least one of said upper and lower membrane retaining members is tapered.
7. An electroelution device for the electroelution and separation of macromolecules from a support, the device comprising at least two chambers, a releasably securable upper membrane retaining member, a releasably securable lower membrane retaining member, and at least one connecting membrane retaining member, at least one of said upper and lower membrane retaining members, each of said chambers, and said connecting membrane retaining member each having a through-hole for passage of a fluid, the chamber through-holes having a diameter that exceeds that of the membrane retaining member through-holes, said chambers defining a membrane well at each end of said chamber through-hole having a sidewall that is provided with a shoulder for receiving a membrane of a prescribed molecular weight porosity and a corresponding one of said membrane retaining members along a common axis such that the membrane is interposed between said shoulder and said membrane retaining member, said sidewalls and said upper and lower membrane retaining members each having complementary-configured engagement surfaces for effecting engagement and retention of the respective membrane retaining members received within their corresponding membrane wells.
8. The device of claim 7 wherein said membranes have molecular weight exclusions such that said macromolecules are retained within the through-holes of said chambers after electroelution.
9. The device of claim 8 wherein said membranes have different molecular weight exclusions.
10. The device of claim 7 which further comprises a holder for holding said support.
11. An electrophoresis tank containing the electroelution device of claim 7.
12. The electroelution device of claim 7 wherein the through-hole of at least one of said upper and lower membrane retaining members is tapered.
13. A process for the electroelution of macromolecules from a support, comprising:
providing an electroelution device having a chamber and upper and lower membrane retaining members releasably securable within membrane wells formed int he chamber, the chamber and at least one of the membrane retaining members defining a through-hole for passage of a fluid, the membrane wells each including a sidewall having a configuration complementary to that of the membrane retaining members to engage and retain the membrane retaining members upon insertion into the wells and a shoulder for receiving an electroelution membrane of a prescribed molecular weight porosity and a corresponding one of the membrane retaining members along a common axis such that the membrane is interposed between the well shoulder and the retaining member; inserting an electroelution membrane into at least one of said membrane wells;
inserting the electroelution device into an electrophoresis tank having a plate, a cathode and an anode;
inserting a support containing macromolecules to be separated into the through-hole of one of said membrane retaining members; and
applying an electric current across said electrophoresis tank to effect electroelution of said macromolecules from said support into the through-hole of said chamber.
14. The process according to claim 13, wherein the electroelution device comprises at least two chambers having membrane wells that are interconnectable by a connecting membrane retaining member, each of said chambers and said connecting membrane retaining member being provided with a through-hole for the passage of a fluid.
15. An electroelution device for immersion in an electrophoresis tank for the electroelution of macromolecules, said device comprising one or more chambers releasably sealable by an upper screw cap and a lower screw cap, each of said chambers and one (but only one) of said screw caps each having a through-hole for the passage of fluid.
16. A device as in claim 15, further comprising a magnet encased within said device for interaction with an external magnet to impart spinning motion to said device.
17. A device as in claim 15 comprising at least two chambers.
This application is a continuation-in-part of application Ser. No. 623,875, filed Dec. 7, 1990 now abandoned.
This invention relates to a device and method for the isolation of macromolecules (including proteins and nucleic acids) from a gel or other polymer supports (or also in solution) to a predetermined volume (10 ul to one ml or more). Furthermore, the macromolecule can be dialyzed and concentrated in the same device.
Electrophoresis traditionally involves establishing an electric field between opposite ends of a solid gel matrix made of agarose and/or polyacrylamide, cellulose acetate. The sample (usually a protein or nucleic acid) is applied on one end of the gel and subjected to electrophoresis. Depending upon various physicalchemical characteristics, the sample migrates through the electrical field at a particular velocity. After electrophoresis, the gel is removed, stained with reagents specific for proteins and/or nucleic acids, destained with organic solvent mixtures and photographed. Whereas electrophoretic separation of macromolecules is an established technique, the elution of macromolecules from the gel is a difficult nonreproducible procedure. Such macromolecules are of potential commercial value because of their applications in science and medicine. Over the last decade or so, various attempts have been made to sluts such macromolecules from gels.
Perhaps the simplest procedure for the elution of macromolecules involves washing the gel and vigorously mixing it with a solution containing salt for several hours. Next, the entire mixture is centrifuged. The macromolecule eluted with the salt solution is precipitated with ethanol. This procedure is very time-consuming and the recovery of macromolecules varies considerably depending upon the nature of the macromolecule. Moreover, ethanol precipitation results in denaturation of several proteins or loss of biological activity of macromolecules.
A similar approach has been taken to elute nucleic acids from agarose gels. Here, agarose gel is melted by heating to 65� C. The mixture is extracted with phenol and the samples eluted. As expected, recoveries are usually low with this procedure. In addition, phenol is a highly toxic and biohazardous substance.
Since diethylaminoethyl(DEAE) cellulose binds deoxyribonucleic acid (DNA), it has been employed to elute DNA from gels. The procedure involves i) electrophoretic transfer of DNA from gels to DEAE-paper. ii) alternatively, DEAE-paper is inserted into slots immediately under each band, thus DNA is transferred electrophoretically. Although these procedures yield excellent recoveries, they are highly dependent on technique and the apparatus is expensive.
Decomposing the gel with chemicals, followed by trapping the macromolecules on glass beads and their elution with salt solution is another method of elution. However, this method is dependent on buffer conditions.
In 1985, Kartenbech introduced an electroelution apparatus (U.S. Pat. No. 4,552,640). This apparatus consists of an upper electrode in the upper chamber and the lower chamber to hold buffer solution and a lower electrode. The upper chamber is separated from the lower chamber by a septum, and the two chambers are connected by a connecting passage within the septum. The end of the lower chamber holds a dialysis membrane, wherein the electrophoretically eluted protein or polypeptide is collected. There are several disadvantages with this apparatus. These are: i) since the volume of the lower chamber is large, it results in dilution of the sample, and ii) since the surface area of the dialysis membrane is large it results in non-specific adsorption of macromolecules resulting in very low recoveries.
In 1985, Walsh introduced an apparatus to sluts nucleic acids (U.S. Pat. No. 4,545,888). This apparatus has features to introduce multiple copies of transfer chamber, filter discs to hold DEAE cellulose and negative electrode. Basically, in this procedure the sample is electrophoresed and collected on DEAE resin (held by a filter disc) at the bottom end of the lower chamber. Next, the filter disc is removed and DNA eluted from the resin employing standard elution protocols. This procedure requires an additional step involving the slution of nucleic acids from DEAE. Moreover, its application to elute proteins and polypeptides is uncertain.
In 1987, Burd introduced an electroelution method and apparatus (U.S. Pat. No. 4,699,706). This apparatus has features in which the electroeluted sample passes through a glass frit and is collected in a semipermeable membrane at the bottom end of the lower chamber. Some of the disadvantages of this apparatus are: i) the dialysis membrane must be held in place by a retaining ring, a gasket and internal shoulders built in the equipment. There are several disadvantages with this equipment. For example, i) this is a rather complex setup and the success depends upon the technique, ii) because the dialysis membrane is smaller then the diameter of the glass frit, it results in poor recovery, iii) use of dialysis membrane results in non specific adsorption of macromolecules, which also contributes to low recovery. iv) there is no possibility of capping the columns to harvest the sample collected in the membrane, v) when the sample cup is removed, it leads to the disruption of the sample collected as it leaks through the filter disc and/or fluid held in the sleeve holding the cup.
In 1986, Clad introduced an apparatus for electroeluting macromolecules from gel (U.S Pat. No. 4,608,147). This apparatus contains an upper chamber which holds a permeable membrane (pore size about 0.2 micro meter) through which macromolecules can migrate downstream. The sample is collected in the lower chamber on top of an impermeable membrane having a molecular weight greater than 1000. Following elution, the polarity of the electric field is reversed for 10 to 15 seconds, so that the macromolecules adsorbed to the inner surface of the outer membrane are released from the membrane into the trap space. There are several disadvantages of this apparatus. These are: i) the use of an impermeable membrane in the lower chamber results in dilution of sample, thus requiring concentration further, ii) because the sample is contaminated with the electrophoretic buffer, an additional step (e.g. dialysis) is required to remove such contaminants.
In 1990, Brautigam and Gorman introduced an electroelution apparatus (U.S. Pat. No. 4,964,961). This equipment consists of a tapered tube divided by a porous disc into an open upper section and a lower section which can be closed by a removable cap. The equipment has a dialysis membrane equal to the diameter of the removable cap and is affixed to it to close off the lower section. After electroelution, the upper section is closed. The sample is collected through the cup and dialysis membrane at the bottom end of the tube. The disadvantages of this equipment are: i) the sample is contaminated and diluted with the electrophoretic buffer; accordingly, it requires dialysis and concentration, further adding to the time effort for such procedures, and ii) non-specific adsorption of sample to the dialysis membrane results in loss of recovery.
In summary, at the present time the separation of macromolecules from a solid phase matrix involves procedures that are elaborate, labor-intensive, expensive, and yields are poor. Such equipment does not have dialysis capabilities resulting in considerable loss of precious macromolecules. In addition, the handling of samples subjects the investigator to a higher chance of contamination with macromolecules. For example, the elution of radiolabeled macromolecules, toxins, and viruses involves electrophoresis resulting in contamination of the apparatus with unbound radioactive material. The eluted protein is usually repeatedly centrifuged against a membrane filter or precipitate in a concentrated salt solution. The resulting material is next dialyzed in a bag which results in removal of salts and at the same time increases the sample volume. Such a sample is next concentrated by ultrafiltration. Taken together, all of these procedures are time-consuming, and often the yields are low (10-30%) compared to the starting material. Furthermore, dialysis and concentration (if required) of the sample are additive steps required to prepare samples, which result in further low recoveries.
FIG. 1 shows an expanded view of one embodiment of the electroelution device according to the present invention.
FIG. 2 shows the chamber of the electroelution device having threads to cooperate with threaded screws in accordance with the present invention.
FIG. 3 and 4 show expanded views of different configurations of the electroelution device of the present invention.
FIG. 5 shows an expanded view of a second embodiment according to the electroelution device of the present invention.
FIG. 6 shows an electrophoresis tank containing the electroelution device according to the present invention.
FIG. 7, 8 and 9 depict further embodiments of the electroelution device in accordance with the present invention.
FIG. 10 illustrates serial interconnection of electroelution apparatus in accordance with the present invention.
FIG. 11 shows electroelution devices having fused chambers in accordance with the present invention.
FIGS. 12 show an electroelution device having a magnet disposed in the chamber thereof in accordance with the present invention.
FIG. 13 shows an electroelution device used in connection with an HPLC valve in accordance with the present invention.
The present invention relates to an electroelution device for the elution of macromolecules from a support. The macromolecules may be nucleic acids, proteins or carbohydrates. The support may be a gel, nitrocelulose, polyvinyl difluoride or the like. Referring to FIG. 1, the chamber 1 is made of any suitable plastic material, including but not limited to plastic or glass. Suitable plastics include Teflon, acrylics and the like. The outer surface of chamber 1 can be conical, round or any other shape. For example, the chamber can be cylindrical for use with a round gasket to prevent leakage. Further, the chamber can have a cubical or rectangular shape to prevent it from rotating or rolling away from a specific position within a tank or other container. The outer surface must be smooth so that it can fit in electrophoresis tank plate 4. It is necessary that a tight seal be made between the chamber 1 and plate 4. This seal can be accomplished by tapered hole, sealing ring or by gasket. The electrophoresis tank plate 4 separates the two electrode (cathode and anode) compartments (FIG. 3) in electrophoresis tank 14. These compartments are filled with the buffer or solution of choice.
Referring additionally to FIG. 1, the chamber 1 has a through-hole 7, which can be of different sizes and diameters. This hole determines the elution volume of the macromolecule. On both sides of the hole 7, there are bigger holes (wells) 3 and 3a, which are tapered at the side. The well surfaces 2 and 2a are smooth so that they can hold membranes 5 and 6. The membranes 5 and 6 can be the same or a different type, such as different materials or a different molecular size exclusion, described below. These membranes can be of different materials, e.g., cellulose acetate, nitrocellulose, PVDF, Teflon, etc. The membrane 6 is semipermeable and holds macromolecules. The small molecules can pass through depending on the molecular weight cutoff of these membranes. Membrane 5 can be a frit or filter membrane, which allows the passage of macromolecules. By using a membrane filter having a particular molecular weight cutoff the membrane will retain macromolecules higher than its molecular weight cutoff. On the other hand, it will allow the passage of molecules of a similar or lower molecular weight than the molecular weight cutoff of the membrane. Membranes 5, 6 can be placed at platform 2 and 2a, respectively, via a tapered screw 9, 9a. The screws 9, 9a and the chamber can be joined, for example, by a threaded fitting (as depicted in FIG. 1) or by a tapered quick-fit or snap-type fitting. In addition, the chamber 1 can be provided with threads 15 on its exterior to cooperate with threads 16 on the screws 9, 9a, as shown in FIG. 2. This configuration is advantageous, for it positions the membranes 5, 6 in close contact with the liquid in the tank to allow for faster interaction.
The screws 9, 9a are tapered to fit the holes 3, 3a, so that they can be screwed into the holes 3, 3a, respectively. Screws 9, 9a are made of the same or a compatible material as the chamber 1. Screws 9, 9a have a smooth surface 10, 10a which fit on the membrane 5 or 6 and tighten on the surface 2, 2a in such a way that no fluid can leak from the hole 7. The leak-proof/tightener can also be achieved by using an O-ring. The membranes 5, 6 have the same diameter as the surface 10 or 10a. By way of example, the volume of the chamber can be as low as 1 microliter to 1 milliliter for analytical purposes, or up to 1 liter or larger for preparative purposes. The diameter of the screw and membrane platform can be from about 0.5 cm to about 10 cm, or larger.
Screw 9 or 9a has a through-hole 8 or 8a, respectively. The hole 8 or 8a can be conical or cylindrical, and can be of different size or shape. The hole 8 serves as a cavity to hold the gel piece or macromolecules on other supports. The holder 11 is made of the same or a compatible material as the chamber 1, and has an outer diameter smaller than the diameter of hole 8 so that it can fit inside the hole 8. This holder 11 can hold the gel piece or membrane, e.g., PVDF (which would otherwise float and can not be retained inside the hole 8 without any support). Screw 13 is the same as screw 9 but does not have a through-hole 8. Screw 13 is used for the closing hole 3 during the dialysis. With reference to FIGS. 3 and 4, the holes 3, 3a and the hole 7 in the chamber 1 can be arranged in various orientations with respect to each other. For example, the holes 3, 3a can be perpendicular (FIG. 3) or coplanar (FIG. 4) and the hole 7 can be disposed at an angle therebetween.
Referring to FIG. 5, an electroelution device is shown which contains multiple chambers 1 and 1a. In this embodiment, a screw 12, in which both ends are tapered, is used to join chambers 1 and 1a. Screw 12 is designed to fit into holes 3 and 3a as are screws 9 and 9a, which are made of similar materials. Screw 12 can join two together two slution chambers. Now membranes 5, 6 and membranes 5a, 6a can have different molecular weight cutoffs and can be inserted in the chamber to separate various macromolecules. In some applications, the gel can be inserted in hole 8 without having membrane 5.
The electroelution device according to the present invention can also be used as a dialysis unit. In this embodiment, membranes 5, 6 are the same and have a molecular weight cutoff lower than the molecule. In this embodiment, screw 13 shown in FIG. 1 is utilized. Furthermore, the elution chamber 1 with membranes can be kept in a vacuum for further concentration of the fluid in hole 7. The chamber 1 will be attached to electrophoresis tank 14 (FIG. 6) by either the angle sealing plate 4, a gasket or with an O-ring.
The chamber 1 can be provided with a very fine, small hole or aperture (e.g. about 1 mm in diameter) at the top portion thereof, as shown in FIG. 7. This hole has certain advantages, e.g. during the application of a high voltage, there is a possibility of the formation of an air bubble, which can stop or otherwise impede the flow of current. The small hole helps to remove the air bubble, and, because the hole dips in the same upper buffer, the biomolecule does not move through small hole due to current in the same direction. The chamber can also be provided with a plurality of openings 18, 19, as shown in FIG. 8. This modification has certain advantages: 1) No air bubble formation; 2) no clogging of membrane as the biomolecule is in circulation; and 3) large volumes of sample can be used without changing the chamber size.
A further advantage of providing the chamber 1 with a plurality of holes 18 and 19 is the capability of providing on-line monitoring of slution or purification of a sample using a detector 22 (e.g. a photometer for protein 280 nm, for DNA 260 nm), as shown in FIG. 9. The conductivity measurement can be used for monitoring for the removal of salts or exchange of buffers. With reference to FIG. 9, the holes 18 and 19 provide interfaces, respectively, to a pump 20 and coding unit 21 which are coupled to the detector 22. The detector, in turn, is coupled to a chart recorder 23 and a fraction collector 24. A power supply 25 is coupled to the electrodes. Finally, multiple chambers 26, 27 and 28 can be used for selective purification, as shown in FIG. 10.
Fused membrane chambers 29 and 30 are depicted in FIG. 11. These chambers are used as disposables, i.e., a new chamber is needed for each application.
With reference to FIG. 12, a magnet 31 can be placed inside the chamber wall in such a way that it has no contact with the liquid. This chamber and magnet arrangement spins when placed in a magnetic stirrer 32 for dialysis or exchange of buffer. The magnet arrangement depicted in FIG. 12 is advantageous because the centrifugal force created by the stirrer 32 and magnet 31 interaction accelerates the exchange of molecules at the membranes 5, 6.
The operation of the electroelution device is illustrated by the following example, which is intended to be only illustrative in nature and not limiting.
FIG. 1 is an exploded view of the entire electroelution apparatus. FIG. 2 shows the joining of the two chambers 1 via screw 12. Gel or membrane holder 11 is employed to hold the PVDF membrane or other gel matrix.
Further objects and advantages of the invention will be revealed from the specifications and the enclosed drawings, which are for illustrative purpose only.
Problem: Elution of a protein of molecular weight 50,000 Daltons, amount 10 ug.
A 100 ul chamber 1 will be taken. A membrane 5 with a molecular weight cut off of 300,000 should be taken. This will serve as the top membrane. The bottom membrane 6 will have a molecular weight cutoff lower than the protein, e.g. 10,000. First put membrane 6 and tighten with screw 9a and then fill the well 7 with the electroelution buffer. Put membrane 5 in such a manner so that no air bubble is trapped in well 7 and close tightly with the screw 9. Fill the wells 8, 8a with the buffer, and then place the whole unit in an electrophoresis tank 14. Both anode and cathode compartments contain 50 mM Tris buffer, pH 8.3. A small piece of gel containing the protein 10�5 mm) is placed in side well 8 (or use holder 11 which can also hold the gel piece or PVDF membrane or other matrix). The electrophoresis chamber lid was closed, current (200 Volts/10 mA) is passed for about one hour. The protein is transferred in to well 7. If required, the Tris buffer can be exchanged by dialysis against a desired buffer by closing the chamber 1 either after replacement of membrane 5 by 10,000 molecular weight cutoff membrane or removal of the membrane 5, and closing using screw 13 which has no through-hole. If the sample is required to be concentrated, the chamber 1 can be put under vacuum.
Problem: PCR Products Purification
Polymerase chain reaction (PCR) is typically used for the amplification of DNA molecules. After the amplification of DNA, the primers are removed by a centrifugation method, by ion exchange or different columns, or by gel electrophoresis. These methods ordinarily require about 60-90 minutes for purification of DNA. The chamber described in accordance with the present invention can be filled with the PCR-reaction product, and at both the ends, a membrane is placed with a molecular weight cutoff larger (MWCO) than the primer of PCR reaction mixture. The whole chamber can be placed in any commercially available electrophoresis tank, without a gasket and with the buffer used for the electrophoresis of DNA. A current is passed for about 5-10 min., the primer is removed completely, and pure DNA is obtained for further sequencing or applications. This method is advantageous because it is fast (5-10 min.), requires only one step purification of nucleic acids (DNA, RNA), (e.g., no centrifugation, column chromatography or precipitation is required), and there is no change in volume. Furthermore, the method allows for high recovery, removes enzymes and salts if proper MWCO is chosen, and is autoclavable. Samples can be concentrated by using two chambers of different size and membranes of different pores. Furthermore, the same method is used for the slution of DNA from Gel, the removal of CsC1 after density gradient purification of DNA, the removal of Ethidium Bromide from DNA sample, and the removal of Gelass or Agragase and hydrolyzed fragment of agarose gel.
Problem: Concentration of Samples
Charged molecules can be concentrated by electrodialysis. Chambers of different volume are used for the concentration of samples. A sample can be placed in large volume chamber, the whole unit assembled as in FIG. 5 and placed in an electrophoretic tank with a gasket. The buffers are used according to application. A current is passed and in such a direction that the biomolecule moves from the larger chamber to the smaller chamber according to the charge of the molecule. Within 5-10 min., most of the sample concentrates in the smaller chamber. By using through flow chambers, a continuous concentration can be achieved. Also, using preparative volume chambers, even large scale samples can be concentrated. Before injecting the sample in HPLC, the sample can be concentrated, desaired, or partially filtered using appropriate membrane or by using through flow cell chambers in combination with an HPLC injection valve. This method can also be used in combination with the autosampler of HPLC. FIG. 13 shows an electroelution device used in connection with an HPLC valve in accordance with the present invention. (FIG. 13.)
Problem: Multiwell Enzymic Reactions
Through flow chambers can be joined in series, as shown in FIG. 10 for the multienzyme reactions for identification of different charged molecules. With reference to FIG. 10, chamber 26 is filled with a sialoglycoconjugate and sialidase, an enzyme which removes sialic acids from sialoglycoconjugates. As soon as the sialic acid is released, it migrates to chamber 27, due to the electric field. Chamber 27 contains another enzyme which cleaves sialic acid to mannosamine and pyruvate. The pyruvate will migrate to chamber 28 where it could be detected by another enzyme followed by a photometer or a biosensor which is placed in chamber 28. By this method, different multienzyme reactions can be monitored in a more specific and selective manner than the multienzyme reactions in one container. Several advantages are realized. For example, different pH--optimums can be used for different enzymes. Therefore, the amount of enzyme used is much lower than the enzyme used in one container, where buffer could be good for pH optimum of one enzymic reaction and not for all enzymes used. Also, the product formed is migrated to another chamber and no auto inhibition is possible. Finally, further purification can be done with affinity chromatography.
Problem: High Salt Barrier (HSB) electroelution:
The invention as depicted in FIGS. 3 and 4 can be used for purification of DNA or proteins by high salt barrier method. This method is patented by Biometra GmbH, Germany. The chamber is generally fixed into a tank. In accordance with the present invention, the chamber can be removed from the tank and is advantageous because it is easy to clean, autoclavable, made of Teflon, and allows high recovery. In addition, different volumes of chambers can be used. Membranes can be used at both the ends or at one end.
The principle objective of the invention is to provide a one-piece apparatus in which to electrophoretically elute macromolecules and later dialyze the sample to any desired volume in the range of 10 ul to ml. Such samples would be free from salts, metal ions, unbound radioactivity and/or other contaminating material. Additional objectives and advantages of the invention are described below.
First, an objective is to provide a gel holder 11 of Teflon or any plastic material that can hold pieces of gel (for example, 10 mm�5 am or any other desirable size) containing the macromolecule and macromolecules transferred to PVDF or nitrocellulose or any other support. Since the amount of macromolecule to be eluted from the matrix is usually small (1-20 ug), the use of holder 11 disallows loss of recovery sample and targets its electrophoretic migration into the well 7.
A further objective is to provide a separator which includes two parts. The top well 8 serves to house the solid phase matrix to which the macromolecules are bound. The screw 9 is separated from the chamber 1 by an inert membrane disc 5 that has a molecular weight cutoff higher than the molecular weight of the macromolecule.
It should be clear from the above that molecular weight exclusion provides at least two advantages: i) the user is free to choose a membrane of any molecular weight cutoff required to elute the macromolecule, ii) if the gel matrix contains other macromolecules (e.g., a western blot containing a protein molecular weight of 50,000 and immunoglobulin G (IgG) molecular weight of about 180,000) then IgG can be retained on the membrane disc whereas the 50,000 molecular weight protein will pass through the membrane resulting in further purification of the 50,000 molecular weight protein.
Another objective of the invention is to provide a chamber 1 for macromolecules into which the macromolecule sample is collected. The advantage of this invention is to provide the user with the option to choose a desired volume in which the macromolecule will be suspended. For example, the user may wish to choose 10 ul to 1 ml as the final volume of sample. We assure the user such capability by providing a chamber 1 precisely to hold 10 ul to 1 ml. This is a very important feature of the invention, since often the amount of macromolecule bound to the matrix is in quantities of a microgram or less. As described above, elution and concentration by dialysis of such macromolecules results in considerable loss of material.
An additional objective of the invention is to pursue dialysis of the macromolecule sample contained in chamber 1. This is achieved by simply unscrewing the screw 9 from dialysis chamber 1. An obvious advantage of this invention is to provide the user with: i) an option to dialyze the sample without transferring into another container (no manual handling required); ii) a choice of buffer against which the sample may be dialyzed that is optimal for the procedures to be subsequently followed, iii) the option to predetermine the volume of the dialysate, for example 10 ul to 1 ml.
An additional objective of the invention is to provide a conventional filter 6 which is capable of entrapping the macromolecules in suspension, but which allows the free flow of molecules with low molecular weight such as sucrose, salt and chemicals (e.g., sodium dodecyl sulphate) and stains such as Coomassie Blue. The advantages of this device are: i) the user has a option of choosing membranes of a wide molecular weight range, resulting in removal of undesired molecules, ii) this membrane device is air-tight disallowing any leakage of the macromolecules. Finally the user removes the macromolecules from the chamber 1 by puncturing the membrane with a fine point needle and withdrawing the sample from the well 7 into a syringe. There are several advantages: i) No cross contamination of the sample with other solution during collection; ii) no manual handling for removal of buffers and membranes is required prior to the collection of the sample; iii) no removal of salts, unbound radioactivity and other contaminating samples is required.
Another objective of the invention is to provide a detachable screw 9, 9a to hold the membrane 5, 6 within the well 3, 3a. The advantages of this device are: i) to juxtapose the membrane 5, 6 against the chamber 1 keeping it air-tight, ii) to allow maintenance of a constant volume 7 within the chamber 1, and iii) the single-edge screw can be replaced with a double-edge screw 13 (FIG. 1). This can be used to attach another chamber 1a to hold membranes and to separate and concentrate yet another protein. Based upon these possibilities, it may be possible to pursue separation and concentration of proteins with grossly different molecular weights.
Another option of the invention is that the slution chamber alone can be used as dialysis unit for purification of macromolecule. However, if further concentration is required, the entire apparatus can be kept in a desiccator under vacuum to achieve the desired concentration.
While the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention, it is to be understood that this disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims.
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Off The Record: On Religion, Politics & Equality
The LA Times reported that the Senate Health Education Labor and Pension committee will have a hearing on child abuse brought on by the horrific sexual abuse scandal at Penn State:
Sen. Barbara A. Mikulski (D-Md.), chairwoman of the Senate Health, Education, Labor and Pension subcommittee on children and families, called the Dec. 13 Capitol Hill hearing, saying that she and others in Congress have been "troubled and distraught about the child sexual abuse allegations" coming out of Penn State. A list of witnesses was being prepared.
There has yet to be a post on the Senate HELP committee website but it will likely come soon.
Although this hearing was spurred by the egregious crimes at Penn State, it is an opportunity to push for a hearing on H.R. 911 which has been introduced anew as H.R. 3126 by Rep. George Miller. H.R. 3126, The Stop Child Abuse in Residential Programs for Teens Act, (READ full text here) would make strides toward regulating not only teen boot camps or juvenile facilities but abusive Christian boarding schools.
H.R. 3126 was introduced on 10/6/2011. It was subsequently referred to the House Committee on Education and the Workforce. Calls to push for a committee hearing can be made here. So far, there is no hearing scheduled. The companion bill in the Senate is S. 1667 (READ full text here) sponsored by Senator Tom Harkin. Harkin, as fate would have it, is the Chair of the Senate HELP Committee (contact info for HELP at bottom of page).
I wrote extensively about this last year, started a petition to get HR 911 a hearing and hand delivered letters and the petition to several legislators. I was contacted by numerous survivors of these abusive Christian homes who were good enough to share their stories with me. Cat Givens and Teresa Frye were the first survivors to share their stories with me in my post History of Violence:
Cathy "Cat" Givens, another New Bethany survivor, shares her experience in an email to me. Cat was at New Bethany from March through December of 1974 during which time she experienced and witnessed her share of abuse. She had her mouth stuffed with soap, accused of lying by Brother Mack Ford, to the point of vomiting. Sick to her stomach, she was then forced to eat dinner – she did so for fear of what may happen if she didn't. Cathy, her gut and mouth still full of soap couldn't help but throw up her meal. For this offense, she was switched on the calves as punishment. This is just one of several humiliations Ms. Givens endured and observed during her time at the home.
Teresa Frye, now a 42-year-old mother of four is a survivor of the New Bethany Home in Arcadia, Louisiana. "It took me 20 years to start talking [about New Bethany] and when I found the message board, I realized what I had been remembering was real."
The survivor websites – Teresa's "message board" – are posted to regularly by former residents of New Bethany and practically every account reads the same: memories of torture, abuse and demoralization at the hands of Mack Ford, Olen King and other New Bethany staff. There are harrowing stories of kids that managed to run away from New Bethany like the one by James, escaping through a Louisiana swamp, and the horrifying account a young man named Guy beaten severely with a golf club.
Teresa was raised in what she refers to as a strict, fundamentalist Baptist church, with "red-faced preachers, hellfire and brimstone," but Teresa says New Bethany was one-hundred times worse.
Before she was sent to New Bethany Teresa remembers Mack Ford and his girls choir performed at her family church. "They were a choir of angels to me, the girls' testimony was so uplifting, I don't know how to describe it," said Frye of her first encounter with Mack Ford, years before she would become a resident at his New Bethany School. A traveling girls choir is nothing new in the Fundamentalist arsenal. Lester Roloff devised a handpicked and disciplined girls' choir as a fundraising and recruiting tool for his original homes.
In addition to singing, the girls gave testimonies — often coached — of their salvation for the hard sell. "They were once drug users and bad kids and going wrong – but they said New Bethany and Mack Ford saved them, we all believed them." Teresa told me this, trying to help me understand how parents could possibly have been fooled and why her parents had no idea what they were sending her into. "Everything looked so good on the outside." Teresa and her friends talk on the message board about their "who had the reddest and blackest butt" contests back in the day.
And they were just the first to come forward. Here are a few comments made by signers of the petitions and survivors of abuse at the hands of these good Christians:
Kim Bowen, Arkansas
As a child I attended Lester Roloff's church in Texas where boys, girls, men and women were housed in non-licensed "christian" group homes. There was an abusive environment, the food was deplorable, and many of these teens were abused mentally and physically. They attended church a minimum of four times a week. They had to enter the auditorium and sit in reserved seating where they were not allowed to talk to anyone but had to keep their eyes averted and read the Bible. These were not bad kids. Just in our religious background you were considered rebellious if you did not go along with the church indoctrination. I believe that many sent to these homes did not need to be there. There must be more accountability in these church "homes."
Dee Rapier, Texas
I at one time taught at the Longstreet Home . We were under Mac Ford . I made many mistakes and have ask tobe forgiven. My x husband was an overbearing person. He was abusive to me and my two boys. He also was abusive the some of the boys. I can only hope and pray that God Has forgiven me. Dee2:21
Teresa Boss Marsh, Oregon
Hello, This issue is very important to me because I a survivor of abuse at an unregulated facility. I attended Victory Christian Academy, in Ramona California in 1989 where I was the victim of abuse along with witnessing daily abuses of others girls during my stay. This was at the hands of a monster who still opens and operated unregulated facilities in Florida and Iowa. He and his wife are prohibited from working with children in the state of California so he has moved his predatory sadistic schools to other states that don't require the facilities to be regulated by the state. Why would anyone oppose state regulation? They are put in place to protect our children. I am 37 years old and it has been over 21 years since my horrible life-changing experience at VCA and I often still wake up form nightmares drenched in sweat as if it were yesterday. For the sake of our children who really are our future, please please I beg you to take this issue seriously and help make this bill a LAW. Sincerely, Teresa Boss Marsh
Jodi Hobbs, California
I have created a website for the girls of Victory Christian Academy who attended either the school in Ramona, San Diego or Jay Florida. Some of the girls have shared their stories on the website. I urge you to take a look. Many still suffer, 20 years later. I wanted these girls to finally have a voice and to feel comfortable to share their experience. I also wanted a place that we all could come together. If you would like to read some of the stories please visit https://girlsofvictorychristianacademy.com
Jonathan Ziv, Canada
Spent two years in the Elan School in Poland Springs, Maine. There was constant psychological abuse and physical abuse. Not sure how the Elan School has been able to stay open all these years when there are so many who have and are willing to tell their stories. For more information about the Elan school visit http://theelanschool.tumblr.com/ or the "I went to Elan school" group on Facebook.
David Hatfield, Oregon
My step daughter went to a therapeutic boarding school. She has nonverbal learning differences and some depression. We wanted to find a school that would help her with these things. The school where we sent her said that they thought they were a good match. After five months we withdrew her from the school because we felt the school was hurting more than helping. If she had reasonable access to unsupervised calls we probably would have withdrawn her earlier. If HR 911 had been in place and we had seen a list of previous violations and fatalities, we would not have sent her in the first place. Thanks for your consideration.
Simone Jones, Kansas
When I was 14 years old, I was in a home called "New Bethany home for girls" I am now 42. I can't even begin to explain the pain and torture I was forced to endure, during my 2 1/2 year stay there. First off, the staff members are not required to pass any type of criminal background check. Where do they come from?? I was abused daily. When I say abused, I mean it literally. Beaten and broken. Left to heal without the care of any medical professional. I had no rights as a human being, let alone a young, scared child. All contact with the outside world was gone. All privacy was gone (yes, even in the restroom, and shower) Not only is physical, emotional and spiritual abuse a problem in these types of places. Sexual abuse is rampant. As children we are told to tell if someone hurts us. Who may I ask are we supposed to tell, if there is no one listening? Children are to be loved, cherished and protected. No battered, broken and forgotten.
And finally, the national media began to take notice. 20/20 ran an expose on Fundamentalist Baptist homes called Scarred Childhood: Whatever Happened to Tina Anderson. Survivors have organized the first annual Survivors of Institutional Abuse convention in February 2012 (information here). Multiple Facebook groups have sprouted up and flourished under the power of the collective survivorship.
There is another petition that you can sign to get H.R. 3126 a hearing and urge its passage. You can also contact your local Senator (link) or Representative (link) and The Senate HELP Committee (link). I also encourage you to contact the individual member of the HELP committee:
Tom Harkin (IA)
Barbara A. Mikulski (MD)
Jeff Bingaman (NM)
Patty Murray (WA)
Bernard Sanders (I) (VT)
Robert P. Casey, Jr. (PA)
Kay R. Hagan (NC)
Jeff Merkley (OR)
Al Franken (MN)
Michael F. Bennet (CO)
Sheldon Whitehouse (RI)
Richard Blumenthal (CT)
Michael B. Enzi (WY)
Lamar Alexander (TN)
Richard Burr (NC)
Johnny Isakson (GA)
Rand Paul (KY)
Orrin G. Hatch (UT)
John McCain (AZ)
Pat Roberts (KS)
Lisa Murkowski (AK)
Mark Kirk (IL)
Additional Reading/Background:
We have too many laws on the books already. We don't need any new ones. We should be demanding that our governors/lawmakers be simplifying our lives with fewer laws, not increasing them. And why in the heck is this being considered at the federal level? This is a state or even district matter. Let the people closest to any problems solve their own problems. Federal government is the least able to decide for the people how they should live.
"Federal government is the least able to decide for the people how they should live."
Oh my god!! What about the victims? Children who are NOT able to decide for themselves but by abusive adults who will beat into them how they "should live". Get your head out of the sand and move on. Something has to be done to abolish these slave institutions and if it takes the Federal government to hear this case, so be it and the Federal government need to do more than requires a mere license to operate these deplorable inhumane prisons camps. These homes violate the constitution of the United States article XIII "Neither slavery nor involuntary servitude, except as a punishment for crime whereof the party shall have been duly convicted, shall exist within the United States, or any place subject to their jurisdiction. Congress shall have power to enforce this article by appropriate legislation."
What goes on in those homes is blantently illegal and this is where christians yell "seperation of church and State, get out of our business". Get it straight, christians made this up, "seperation of church and State" for the church to abuse authority. The Declaration of Independence was created for we the people who have unalienable rights to pursue life, liberty and happiness and that includes children and some adult christians abuse this authority under the banner of "seperation of church and State", sick.


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Activities and Amenities
|Within Facility
|Within 10 Miles
Public Workshop Notice: BRANSON, Mo. – The Army Corps of Engineers is hosting the Table Rock Lake Shoreline Management Plan scoping workshops March 26-28 to share information about the update process and to collect public comments concerning shoreline activity around the lake. Anyone interested in the future of shoreline activity use around Table Rock Lake is invited to drop in anytime during the workshops. The scoping workshops will be held at the Branson Convention Center, 200 Sycamore Street in Branson from noon until 4 p.m. Thursday, March 26; 4 p.m. until 8 p.m. Friday, March 27; and noon until 4 p.m., Saturday, March 28. For more information go to http://go.usa.gov/MG34.
part ofTable Rock Lake, US Army Corps of Engineers
Status: Closed through Tue Mar 31 2015 Season Dates
OverviewOld Highway 86 is a spacious and sunny campground that sits along the White River Arm of Table Rock Lake in the rolling Missouri countryside.
Natural Features:
Table Rock Lake is a beautiful body of water surrounded by a forest of oak and hickory trees. Its waters wind down through the valleys and hollows of the Ozark Mountains, from Branson, Missouri to Eureka Springs, Arkansas.
Whether on water or land, Old Highway 86 Campground offers visitors countless recreational activities. Picnicking, swimming, scuba diving, boating, fishing, hunting, hiking and wildlife viewing are all popular pastimes at the park.
Old Highway 86 Campground offers 71 reservable campsites, along with one large reservable group picnic shelter. Amenities like hot showers, drinking water, a dump station and electric hookups create a comfortable camping experience. One swimming area, a boat ramp, playground and volleyball court make the campground perfect for family trips.
Nearby Attractions:
The Dewey Short Visitors Center is located at the south end of Table Rock Dam. It offers three educational films focused on the history of the dam, the story of Lewis and Clark and the history of eagles and their endangerment in the state of Missouri.
Activities and Amenities
Know Before You Go
Getting There:GPS Info. (Latitude, Longitude):
36.55944, -93.31944
From Branson, Missouri, take Highway 65 and turn west onto Highway 86. After 6 miles turn north on Highway UU and follow signs to the campground.
Contact Information:
Mailing Address:OLD HIGHWAY 86
1791 State Hwy UU
Phone Number:Information: (417)779-5376
Additional Information
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I checked with some friends at work that have TWC and I guess TWC's whole house DVR absolutely blows chunks. Each "satellite" DVR in your house is capable of recording 2 shows at once. Looks like WOW's WHDVR setup has nothing to fear from TWC's WHDVR. Two of the guys I spoke to switched to satellite a while back because of TWC's DVR limitation (can only record 2 shows at once) and their crappy DVR software. (Constant lockups mainly.) In my eyes, the only thing TWC has going for it then is the app that lets you watch the live TV package your subscribed to on your iPad/Android device. A 3rd person I asked, dumped all TWC video services and only has them for internet because their prices were skyrocketing.The more I think about it, the more I like the idea of just using my ISP for internet. The only thing on TV is crappy, "reality" shows..........on EVERY. SINGLE. CHANNEL. God, this scourge of "reality" TV can't end soon enough. I'm working on breaking my wife of her "reality" TV addiction and once that's done.......... internet only package please!
"TWC can already do 1TB worth of storage and 4 tuners on their signature home service in which they tie two multiroom DVRs together for a total of 4 tuners and 1TB worth of storage. I think they have an intro deal for Signature home for $199 that includes the multiroom setup, basic digital tier, 50Mbps internet (75Mbps in Dallas area)(100Mbps in Kansas City area),and digital phone)"


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Nike Air Sport Stand Bag
$112.49 $149.99*
Save $37.50 (25%)
Product Information:
Gear up for your next round with the Nike® Air Sport Stand Bag. This athletic stand bag features an 8-way top with full-length dividers for easy transportation and club storage. 9 total pockets, including a water-resistance, fleece-lined valuables pocket and insulated cooler pocket, provide you with enough storage space for the essentials. Nike® EquaFlex® Max Air revolving double straps makes carrying your bag easy and comfortable.
- Nike® 2014 Air Sport Stand Bag
- 8-way top with grip handle and full-length dividers
- 6 zippered pockets, 9 total pockets for item storage
- Includes fleece-lined valuables pocket and insulated cooler pocket
- Nike® EquaFlex® Max Air curved revolving double straps
- Integrated GPS loop, pen sleeve and glove patch for convenience
- Ventilated air mesh back pad for increased carrying comfort
- Custom leg end caps provide a sturdy structure on any surface
- Includes matching rain hood for use during inclement weather
- Nike
Country of Origin: United StatesWeb ID: 24985486
Customer Reviews:
Nike Air Sport Stand Bag 26 26
This product is very easy to use I brought this for my husband and he loved it! He couldn't stop talking about it to his gulf buddies. There were a lot of nice features to this bag & he loved the various compartments. January 4, 2015
Very nice bag Lots and lots of pockets. Was really light. Overall very please January 2, 2015
Great purchase Bought this bag for our son for Christmas. He loves it. Many nice features including good storage pockets and comfortable shoulder straps. December 27, 2014
Gift for my son! This golf bag was a birthday gift for my son. I have not seen the bag as it was delivered straight to his home in California within 3 days from being ordered online. Impressive. He called and loves the bag. Lightweight, as he loves to walk rather than ride. October 28, 2014
Light, well constructed, best quality Love the bag. Looks sharp and carries everything I need comfortably. October 27, 2014
Great golf bag with style Have used this bag two times and I love it. Super comfortable to carry and lots of great pockets. Especially like insulated drink holder keeps your beverages cold. Really comfortable to wear if you are walking the course, and multiple cool color options. September 15, 2014
Very Pleased Works great for carry or cart. Comfortable to carry and is designed well. I received a Maxfli 5.0 as a gift and returned it due to being uncomfortable to carry. Glad I traded up to this bag. Fully contained lined water bottle holder will hold two bottles of water and keep them secured. September 6, 2014
golf bag Generally the golf bag is very good. The legs for the stand are a bit weak. August 12, 2014
Nice Carry Bag Bought this for my 12 yr old son......its light and stylish! August 12, 2014
Love my new bag! So comfortable with tons of pockets and lots of compliments and room for clubs. August 6, 2014


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U.S.-based chip maker Broadcom, whose Wi-Fi and Bluetooth combination wireless chips are found in such popular smartphones as the Samsung Galaxy S III and Apple iPhone 5, has announced NFC support for one of its next wireless combo chips.
Nintendo has released its much-anticipated Wii U gaming console with NFC technology, enabling the game maker to incorporate physical objects into game play, in addition to possible support in the future for contactless payment for digital content.
Struggling video game maker Nintendo will incorporate NFC in its planned Wii U home video game console, including support for payments, the company's president Satoru Iwata said today.
NFC Times Exclusive Insight – A U.S. district court judge has ruled that T-Mobile USA infringed an NFC patent held by Israel-based On Track Innovations.
NFC Times Exclusive Insight – France-based Gemalto, which is keen to take the spotlight off its SIM business, projects EMV cards to represent perhaps the largest expected source of revenue growth in the coming year.
NFC Times Exclusive Insight – UPDATED: Mobile point-of-sale device supplier Square has acquired payment processing component vendor Kili Technology, the same vendor that purchased NFC accessory vendor DeviceFidelity in November, but Square does not plan to keep DeviceFidelity, a source connected with Square told NFC Times.
NFC Times Exclusive – NFC Times has learned new details about how Samsung intends to make money from its planned payments service, Samsung Pay; as well as the technology architecture the OEM will use and the identity of two unannounced banks, along with a new category of issuer that is planning to participate.
NFC Times Exclusive – The public relations debacle Gemalto faced following revelations last month that U.S. and UK spy agencies had stolen keys from its SIM cards is not the only problem the France-based vendor has had to deal with regarding SIMs.
NFC Times Exclusive – MasterCard Worldwide has named James Anderson group executive for platforms, a key position inside the payment network, which is seeking to secure its role in the ecosystem as mobile payments start to gather steam, NFC Times has learned.
NFC Times Exclusive Insight – One of Australia's largest banks, Commonwealth Bank, is launching NFC payments from its mobile-banking app using host-card emulation, having earlier tried embedded secure elements, contactless stickers and iPhone attachments.
NFC Times Exclusive Insight – Google's recently announced API for NFC payments applications, Android Pay, is expected to support apps using host card emulation, or HCE, but also likely will support storage of keys on hardware.
NFC Times Exclusive Insight – Apple's new wearable, Apple Watch, will support Apple Pay as well as mobile keys for hotel rooms, the company announced Monday.
BARCELONA, Spain – NFC Times Exclusive (UPDATED): The Galaxy S6 will carry an embedded secure element, mainly to enable nonpayment applications, while supporting NFC payments with host-card emulation and storing payment tokens in a secure area on the processor, an ARM-based TrustZone, a representative of Samsung told NFC Times. The embedded secure element also could be used to store tokens outside of the U.S.
BARCELONA, Spain – NFC Times Exclusive Insight: Samsung Electronics has unveiled its Apple Pay rival, which it calls Samsung Pay, a mobile-payments service that, as expected, supports technology from Samsung's recent acquisition of LoopPay combined with NFC.
NFC Times Exclusive Insight – Following a lull in attention on host-card emulation with the announcement and launch of Apple Pay in the latter half of last year, the buzz appears to be back on HCE, and among the latest organizations promoting it is MasterCard Worldwide.


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You can now invite anyone to meet at your favorite Starbucks.
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29 year old woman | Greenbrae, CA, USA | Seeking men 28-40 within 30 miles
Sweet & playful, looking for someone kindhearted & funny
I am a Bay Area transplant: I moved here 2.5 years ago from Chicago for a job in San Francisco that I truly love. Work is where I invest much of my time and energy during the week. My office is in the financial district of SF. I commute from Marin, usually on the ferry, which is pretty much the coolest possible way to get to work. A few months ago, I adopted a sweet little Chihuahua named Lizzie. She keeps me active and entertained, whether we're going for long walks around beautiful Marin, or whether she's burying doggie treats in my laundry! Lizzie is a wonderful companion. I am passionate about food and wine. A few of the specialties in my recipe box include crab cakes, linguine with spicy sausage & arugula, and fried chicken with mashed potatoes & gravy. I like to keep my wine rack stocked with a couple interesting bottles of wine. I also love to listen to good music while I cook; I am a sucker for the singer-songwriter types, like Ray LaMontagne, Ryan Adams, and John Mayer. I am an eternal optimist. I consider myself an easy person to be around. I mix sweetness with playful sarcasm. I listen more than I talk, though I do not hesitate to speak my mind. I am honest and straightforward, with a solid set of values. A quote that I try to live by is, "Always do right. This will gratify some and astonish the rest." (Mark Twain) So what interests me in a match? - Kindness is important to me. This is a trait that is not often mentioned, yet it translates in so many ways in a person's character and behavior. - Humor is required. If we cannot laugh together (or at each other), where will the fun be? - Intelligence & depth, so we can share meaningful conversations. - Work ethic. I work really hard at my job, and I need to respect the other person's work ethic, too. - Self-awareness. It is important to know who you are and what makes you tick. - Passion. Two meanings: first, to be passionate about at least one thing in your life (work, a hobby, etc.), and second, to be passionate in relationships, as well. Life is a journey, and it is better with good company.
Coffee and conversation , Cooking , Dining out , Movies/Videos , Music and concerts , Exploring new areas , Performing arts , Religion/Spiritual , Travel/Sightseeing , Wine tasting
Walking / Hiking
I exercise 3-4 times per week.
I have Dogs
I have a sweet-tempered, 9-lb, 1.5 year old Chihuahua named Lizzie. She is such a joy!
Middle of the Road
I work in San Francisco and live in Marin. I love exploring both. Anywhere with good food is where I want to be.
Cornell University, Ithaca, NY
Music: Ray LaMontagne, Tristan Prettyman, Brett Dennen, Ryan Adams, Adele, Lady Gaga. TV: Breaking Bad, Californication, terrible stuff like The Bachelor. Movies: Crazy Heart, No Country for Old Men, The Social Network, It's Complicated.
I am an up-for-anything type of gal who will try anything at least once. I have just as much fun going out on the town as I do staying home and sharing great conversation.
"This Is How You Lose Her" by Junot Diaz and "Quiet: The Power of Introverts in a World That Can't Stop Talking"
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'5" (165cms)
|5'5" (165cms) to 6'5" (195cms)
|Body type:
|About average
|No preference
|No preference
|Light brown
|No preference
|No Way
|No preference
|Social Drinker, Regularly, Moderately
|Sales / Marketing
|No preference
Brand Direct Marketing
|I'll tell you later
|No preference
|Never Married
|Never Married, Widow / Widower, Divorced
|Have kids:
|Wants kids:
|Definitely, Someday
|White / Caucasian
|White / Caucasian
|Christian / Protestant
|Christian / Catholic, Christian / Protestant, Spiritual but not religious, Christian / Other
My faith is important to me, but I do not impose it on others.
|Bachelors degree
|Bachelors degree, Graduate degree, PhD / Post Doctoral
Major: Hotel Administration


text:
Hot Springs Village News
Local news for Hot Springs Village, AR continually updated from thousands of sources on the web.
National Town Builders Association roundtable convenes this weekend
Wednesday Mar 18 | Log Cabin Democrat | Comment?
Thor spews sleet, snow into central Arkansas.
Thursday Mar 5 | CNN | Comment?
Updated: Fri Mar 20, 2015 04:30 pm
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Fantastic Savings with Voucher Codes BED015 or BED030 or BED040
Bedworld are proud to offer voucher codes to all our customers. The voucher codes can be used towards purchases on our online bed shop. When you are in the Bedworld basket, simply enter one of our bed mattress voucher codes which will automatically deduct voucher discount off your total order. The codes can be used on all items on Bedworld including our great selection of beds, mattresses and bedroom furniture. So go on, treat yourself and have a reward from us.


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A Long Walk to Freedom: Early Years, 1918-1962 v. 1: 1918-1962Paperback
List price $14.83
You save $6.71 45% off
Free delivery worldwide
Dispatched in 1 business day
When will my order arrive?
- Publisher: Abacus
- Format: Paperback | 512 pages
- Dimensions: 108mm x 174mm x 40mm | 259g
- Publication date: 21 November 2002
- Publication City/Country: London
- Illustrations note: Section: 8, b/w
- Sales rank: 9,559
Product description
The riveting memoirs of the outstanding moral and political leader of our time, LONG WALK TO FREEDOM brilliantly recreates the drama of the experiences that helped shape Nelson Mandela's destiny. From his beginning in the Transkei to his being taken to Robben Island, this is the remarkable story of how a man rose so far, only to be sentenced to life imprisonment. Emotive and compelling, this is the story of an epic life. 'Burns with the luminosity of faith in the invincible nature of human hope and dignity ...Unforgettable' ANDRE BRINK 'Enthralling ...Mandela emulates the few great political leaders such as Lincoln and Gandhi, who go beyond mere consensus and move out ahead of their followers to break new ground' Donald Woods in the SUNDAY TIMES
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Author information
Nelson Mandela became the democratically elected, first black president of the republic of South Africa on 27 April 1994. He died on 5 December 2013.
Review quote
A tale of anger and sorrow, love and joy, grace and elegance DAILY NEWS Indispensable ... [a] unique life-story Anthony Sampson A truly stunning account of his extraordinary life ... A vivid testimony to an unusual mixture of courage, persistence, tolerance, and forgiveness Sir David Steel One of the most life- affirming books you'll read GQ


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Spiga has good food and service. Spiga came highly recommended to my girlfriend and I and while we were happy with the restaurant we thought it was not up to the quality we received at other restaurants. Spiga is up the street from most of the restaurants of Grand Case.
If you own or manage Spiga, register now for free tools to enhance your listing, attract new reviews, and respond to reviewers.


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|Small Greeting Card
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NIKON D300s
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Oh yes! Beautiful lines and patterns.
Very well percieved. Great capture as usual.
Cheers Jim
Excellant shot, well titled!
Brilliant! LOVE it!
Great find & capture..great find & capture..
Thanks a lot dear Andrew.
– Jean-Luc Rollier
great capture!
a great black and white image
Very nicely seen Jean-Luc… ;-)
fantastic, love it!


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VIDA: Women in Literary Arts recently released several large pie charts comparing how women and men are published in some of the largest literary magazines—who's reviewing books, whose books are being reviewed, and who's being interviewed. Out of 40 charts, women outnumbered men on only two of them.
A piece of advice for those who are interested in reading Am I Blue?, an anthology of YA short stories about gay, lesbian, and questioning characters: don't read it on the bus. I tried several times, and always missed my stop by many blocks without fail. The collection is engrossing to a plan-canceling degree; editor Marion Dane Bauer has put together funny, poignant, compassionate, impassioned stories about teenagers supporting each other, by authors dedicated to both advocacy and entertainment.


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Find commute times to your work, school, gym and more.
Astoria at Hallet's Cove
Contact this property
Floor Plans
Prices, specials, features and availability subject to change.
Business Center, Clubhouse, Disability Access, Elevator, Extra Storage, Full Concierge Service, On Site Maintenance
100% Smoke Free Living, 24-Hour Attended Lobby, 7th & 8th Floor Entertaining Terraces, BBQ/Picnic Area, Bicycle Storage, Efficient Appliances, Green Building, High Ceilings, Individual Heating & Cooling Units, Manhattan Skyline Views*, Recreation and Media Room, Residents-Only Gym with Free Weights, Rooftop Lounge Featuring: Cabanas, Chaise Lounges, Symmons Faucets & Fixtures, White Quartz Countert, Upgraded Kitchens with Frigidaire SS Appliances
Property Details
Welcome to Astoria at Hallet's Cove!!!
Astoria at Hallet's Cove is located steps away from the Astoria waterfront with landmarks such as the Socrates Sculpture Park, The Noguchi Museum and Rainey Park and is just a short distance to public transportation and everything the neighborhood of Astoria has to offer. Astoria at Hallet's Cove offers its residents modern boutique living with luxurious amenities and exceptional NYC skyline views.
Leasing Terms
Lease terms are variable. Please inquire with property staff.
Pet Policy
Dogs Allowed. Cats Allowed. Pets Accepted. Call for service animal policy.
Community Information
79 units
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Bees in Yard
I'm having a bit of a problem here, ok, not a bit, a large problem. Here is some backgroud. Since June of 2004 some bees have been flying in and out of the underneath of a metal shed in my yard. The metal shed is sitting on top of some cement bricks. The cement blocks might be around the perimeter of the shed and not all the way across the underside, so that there is plenty of unobstructed space underneath. Some of the bricks may have spaces in between, due to misalignment, and I believe some are just broken in some areas, enough to where the bees have been able to enter.
I placed some dirt and mud next to one side of the shed when I saw the bees trying to enter there. They later moved to the back side of the shed and that area is slightly less accessible. I had used up much dirt to cover the side bricks and so by the time I tried to dig up more to cover the back of the shed, the bees had really began to become established. I didn't want to throw dirt on the entrance while they had become increasingly active, but I now realize that they have established themselves so well in our yard, that I can no longer deal with having them there. I have a dog that has had to stay indoors lately because the bees have been staying on trees and I don't want him near them. I saw a huge swarm in our yard in June of 2004 and one yesterday April 25,2005. There was another smaller swarm that I saw about a week ago. Each of these times I was outdoors, and luckily was able to get myself and my dog indoors safely.
The questions in my mind right now are as to whether I should cover up the area behind the shed, hope the bees that are outside of it move on, and then remove the shed and whatever might be underneath (including the bricks),or can someone look underneath just as is (with live bees underneath)? I wanted to go the route of having a live bee removal, but being that these are probably considered "wild" bees, they wouldn't be of use to keep around or am I mistaken? The swarming and now having these bees on our trees that aren't leaving is distressing. Can anyone advise me on what to do?
As you don't seem to know what type of be you're dealing with, you should be cautious dealing with bees. You should try to find a beekeepers club or organization in your area or call an exterminator who can deal with bees. A beekeeper may, or may not, be willing to deal with your bees if they're honeybees. If they're not, beekeepers really have no use for them, and the exterminator will be your best bet.
Hi Treebeard. I appreciate your input. I spent about a week trying to decide what to do about the situation. These bees never seemed to pose a direct threat to myself or anyone else. Over the last several months I would spend time outdoors and observe the bees at a fairly close distance collecting pollen from the flowers in our yard. Their docile nature led me to believe that they were an asset to the environment. I assumed they were non-hostile honeybees.
The thing that had me most concerned was seeing the large quantities of bees spending time on trees and the swarming prior to and afterwards. In one weeks time there were three swarms in total and I observed two of them. The first one I didn't see, but I did see my dog under the tree where the bees had gathered.
The shed is about ten feet by five. Yesterday we had some people remove the bottom of the shed in pieces (with electric tools) and I saw the honeycomb being removed. Live removal had been something I considered, but the size of the shed played a role in our ability to access the bees and safety was also a concern.


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Rogers 50th anniversary
David and Lois Rogers, Wilmont, will celebrate their 50th wedding anniversary with an open house from 2 to 4 p.m. Saturday at the Wilmont Community Center.
They were married Dec. 3, 1960, in Dell Rapids, S.D.
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1. Peel tangerines reserving half of the peel.
2. Remove white membrane and seeds.
3. Then chop the tangerines coarsely.
4. Wash cranberries drain and remove stems.
5. Put cranberries and reserved peel through coarse blade of food chopper.
6. Add tangerines and sugar and mix well.
7. Refrigerate for about 2-4 hours and serve with garnish as desired.


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Information Without The Bun
Not your stereotypical librarian
By Roger Green | About this blog
Posts filtered on Category
Movie review: The Grand Budapest Hotel
The Grand Budapest Hotel Is a cleverly oddball screwball comedy caper, yet melancholy tale of murder, [Read More]
Movie Review: The LEGO Movie
I'm still uncertain that The LEGO Movie was designed for children. [Read More]
50th anniversary of SELMA: see the movie January 6, support the Underground Railroad History Project
Join with other supporters of the Underground Railroad History Project for the benefit Special Advance Screening of SELMA, scheduled for Tuesday, January 6, 2015. [Read More]
37th Annual Kennedy Center Honors
I'm sure I first heard The Police on WQBK-FM, my favorite radio station in 1979. [Read More]
Mike Nichols
I found out about Mike Nichols' death because my TV was possessed. [Read More]
Sophia Loren is 80
Sophia Loren was the first performer to win an Oscar for a foreign-language performance. [Read More]
Frank Oz is 70
Frank Oz directed films such as The Muppets Take Manhattan (1984), Dirty Rotten Scoundrels (1988), What About Bob? (1991), Housesitter (1992), and In & Out (1997). [Read More]
George Lucas is 70
Every Star Wars Fan Complaint About Episodes I through III In A Music Video Parody [Read More]
Movie recasting, and race
Remakes have often meant casting black actors in roles where the source material suggested a white character. [Read More]
My Academy Awards guesses
If the Pope were voting, it might be for Judi Dench. [Read More]
Show More


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Dissolve the Palestinian authority!
Mazin Qumsiyeh writes: In my last email, I asked readers what should Palestinian leaders do now that the Pal Authority is clearly without authority and is becoming bankrupt and Oslo process was shown a scam and can never lead to peace. 112 individual readers responded and not one said to stay the course.Sumud … »


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Use a combination of these core back exercises to strengthen your lower, upper or middle back areas. Whether you're interested in barbell deadlifts or reverse extensions, we have back strengthening exercises for every level. These exercises are also featured in convenient workout routines that include proper warm-up and cool-down techniques, which greatly reduces the risk of injury.
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Injuries occur at any time and for any reason. If you get injured, you may not know how to proceed and prepare for exercise post-injury. This mind-and-body guide can help prepare you for a brighter, injury-free future in fitness and performance.
Do you experience chronic pain? It can be debilitating and limit you from living a full life. However, overcoming or managing pain is often achievable through a mind-body approach. Learn how you can better manage your pain by employing one or more mindful-based exercises...
Eating healthier can be tough, but that task becomes extremely difficult when temptations from within the kitchen seem to beckon you. Squash those diet-breakers with these five strategies to help you stay focused on your goals ahead.
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