text:
Double stranded nucleic acid is denatured by subjecting a solution thereof to a voltage applied between electrodes spaced by no more than 1.5 mm in a time not previously achievable in electrochemical denaturation. PCR is practiced isothermally by periodic application of voltage to produce denaturation. Electrochemical cells and kits for use in the process are provided.
1. A process for denaturing double-stranded nucleic acid which comprises subjecting a solution containing said nucleic acid to a voltage applied between electrodes under conditions such as to convert at least a portion of said nucleic acid to a wholly or partially single stranded form in the solution, wherein said electrodes approach to within 1.5 mm of one another in said solution.
2. A process as claimed in claim 1, wherein the electrodes approach to within 1 mm of one another in said solution.
3. A process as claimed in claim 1, wherein the electrodes approach to within 0.5 mm of one another in said solution.
4. A process as claimed in claim 1, wherein a voltage of from 0.5 to 3 volts is applied between said electrodes.
5. A process as claimed in claim 4, wherein a voltage of from 1.5 to 2.5 volts is applied between said electrodes.
6. A process as claimed in claim 1, wherein the solution contains a promoter which assists said denaturation.
7. A process as claimed in claim 6, wherein said promoter is methyl viologen or a salt thereof, or is a multivalent inorganic cation.
8. A process as claimed in claim 7, wherein said promoter is magnesium or lanthanum ions.
9. A process as claimed in claim 1, wherein where the electrodes most closely approach one another, one or both of the electrodes is pointed.
10. A process of repeated denaturation of double-stranded nucleic acid, wherein the nucleic acid is denatured by a process for denaturing double-stranded nucleic acid which comprises subjecting a solution containing said nucleic acid to a voltage applied between electrodes under conditions such as to convert at least a portion of said nucleic acid to a wholly or partially single stranded form in the solution, wherein said electrodes approach to within 1.5 mm of one another in said solution, and in which said voltage is applied as a repeating pulse having a duration of up to 2 minutes.
11. A process of repeated denaturation as claimed in claim 10, wherein said voltage is applied as a repeating pulse having a duration of up to 1 minute.
12. A process of repeated denaturation as claimed in claim 10, wherein between said pulses the voltage is turned off or reversed for a period equal to the period for which the voltage is applied.
13. A process of repeated denaturation as claimed in claim 12, wherein said voltage is applied as pulses at a frequency of from 1 to 100 Hz.
14. A process of amplifying a target sequence of nucleic acid comprising hybridisation, amplification and denaturation of nucleic acid wherein said denaturation is conducted by subjecting a solution containing said nucleic acid to a voltage applied between electrodes for a period of up to 2 minutes under conditions such as to convert at least a portion of said nucleic acid to a wholly or partially single stranded form in the solution.
15. A process of amplifying a target sequence of nucleic acid as claimed in claim 14, wherein said electrodes approach to within 1.5 mm of one another in said solution.
16. A process of amplifying a target sequence of nucleic acid as claimed in claim 14, wherein said voltage is applied as a repeating pulse having a duration of up to 2 minutes.
17. A process of amplifying a target sequence of nucleic acid as claimed in claim 14, wherein said voltage is applied as a repeating pulse having a duration of up to 1 minute.
18. A process of amplifying a target sequence as claimed in claim 14, which is a PCR or LCR amplification.
19. An electrochemical cell for use in the denaturation of nucleic acid comprising a well having an electrically conductive inner surface constituting a first electrode and a second electrode dipping into said well and reaching to within 1.5 mm of the floor of said well.
20. A kit for use in a nucleic acid assay or amplification, comprising an electrochemical cell as claimed in claim 19, and at least one of:
a nucleic acid for denaturation;
an oligo-nucleic acid;
one or more nucleotides;
a polymerase;
a ligase; and
a buffer solution.
21. An electrochemical cell for use in the denaturation of nucleic acid comprising a well having a floor with an electrically conductive inner surface which constitutes a first electrode, and a second electrode separate from the well which dips into the well and reaches to within proximity of the floor of the well.
22. The electrochemical cell of claim 21, wherein the second electrode is adjustable in height with respect to the floor of the well so as to reach to close proximity to the floor of the well.
23. An electrochemical cell for use in the denaturation of nucleic acid comprising a plurality of wells, each said well having an electrically conductive inner surface constituting an electrode, said plurality of wells forming a first array of electrodes, and a second array of electrodes separate from said first array of electrodes, wherein at least one electrode of said second array of electrodes dips into at least one well of said plurality of wells so as to reach to close proximity to the electrically conductive inner surface of said at least one well.
This application claims benefit of international application PCT/GB95/00542, filed Mar. 14, 1995.
This invention relates to processes for the treatment of nucleic acid material in order to effect a complete or partial change from double-stranded form to single-stranded form and to processes of amplifying or detecting nucleic acids involving such denaturation processes.
Double-stranded DNA (deoxyribonucleic acid) and DNA/RNA (ribonucleic acid) and RNA/RNA complexes in the familiar double helical configuration are stable molecules that, in vitro, require aggressive conditions to separate the complementary strands of the nucleic acid. Known methods that are commonly employed for strand separation require the use of high temperatures of at least 60� C. and often 100� C. for extended periods of ten minutes or more or use an alkaline pH of 11 or higher. Other methods include the use of helicase enzymes such as Rep protein of E.coli that can catalyse the unwinding of the DNA in an unknown way, or binding proteins such as 32-protein of E.coli phage T4 that act to stabilise the single-stranded form of DNA. The denatured single stranded DNA produced by the known processes of heat or alkali treatment is used commonly for hybridisation studies or is subjected to amplification cycles.
Such separation is a prerequisite of a number of protocols involving the in vitro manipulation of nucleic acids, one example of which is a reaction that produces multiple copies of target sequences of DNA and which employs a heat-stable polymerase enzyme (U.S. Pat. No. 4,683,202, K. B. Mullis et al). This development, known as the polymerase chain reaction (PCR), is of significant commercial importance and strand separation is normally effected by heating the sample to approximately 95� C. The removal of the need to heat the sample would provide a number of benefits. For example, it allows the design of compact and readily controllable apparatus, and the use of higher fidelity mesophilic enzymes.
WO 92/04470 discloses a process whereby nucleic acid strands are separated by the application of an electric field. The advantages of the electrical method are discussed in greater detail, along with the method's application in amplification reactions such as PCR and ligase chain reaction. Forms of electrochemical cells for carrying out the reaction are described and also the use of "promoter" compounds that enhance the efficiency of denaturation.
Prior to WO92/04470, a number of other workers had described denaturation of DNA in electrochemical cells. However, in none of these cases was single-stranded product left free in solution in useful quantities. Rather, DNA appears to have become irreversibly bound to the surface of the electrode, in which condition it is not available for further participation in processes such as PCR. In the method of electrical denaturation described in WO92/04470, single strands accumulate in solution and their utility and integrity is confirmed by subsequently performing PCR.
In WO92/04470 electrical denaturation of DNA was carried out using an electrode comprising a central rod of glassy carbon encased in a teflon sleeve except at its end. The working electrode was of platinum mesh lying against the teflon sleeve. A calomel reference electrode was used, situated in a side chamber which was connected to the main cell by a capillary tube (see Stanley C. J. et al, J. Immunol. Meth. [1988], 112, 153-161). Using this apparatus the most rapid denaturation was achieved in 15 minutes with the working electrode at a potential of -1V with respect to the reference. The presence of NaCl in the reaction delayed denaturation.
In WO92/04470, a PCR reaction is conducted in which there are repeated denaturation operations conducted using the electrochemical cell described with intervening amplification stages. The denaturation stages are each conducted for a period of five minutes or longer and the total time for the PCR reaction is therefore very extended. Furthermore, the conditions under which the PCR reaction was conducted electrochemical in WO92/00470 differ from those of the conventional PCR process in that it was not found possible to use a conventional PCR buffer system. In order to obtain denaturation, it was necessary to conduct the process at a much lower ionic strength than would be consistent with such a buffer system. Excluding the promoter methyl viologen, the process was basically conducted in distilled water.
It has now been discovered that it is now possible to conduct a denaturation electrochemically considerably faster than is disclosed in WO92/04470 and to conduct an amplification procedure much faster than is disclosed there.
Although the spacing between the two working electrodes in WO92/04470 is not explicitly stated, it was in fact several millimetres.
Accordingly, the present invention provides a process for denaturing double-stranded nucleic acid which comprises subjecting a solution containing said nucleic acid to a voltage applied between electrodes under conditions such as to convert at least a portion of said nucleic acid to a wholly or partially single-stranded form in the solution, wherein said electrodes approach to within 1.5 mm of one another in said solution. Preferably, the electrodes approach more closely, e.g. within 1 mm or more preferably 0.5 mm of one another. Ideally, the electrodes are adjusted to be spaced by as little as possible whilst ensuring that they do not contact one another to produce a short circuit.
It is preferred to apply a voltage difference of from 0.5 to 3 volts between the electrodes. Voltage differences above 3 volts seem to inhibit denaturation although the mechanism involved here is presently unknown.
Preferably, the process is conducted at a voltage of 1.5 to 2.5 volts measured as a voltage difference between the electrodes.
Preferably, where the electrodes most closely approach one another, one or both of the electrodes is pointed. Such an electrode may be provided with a single point or a plurality of points. There appears to be some interrelationship between the ideal voltage applied and the shape of the electrode and it may be that there is a preferred or ideal field gradient at the point of the electrode which can be achieved by adjustment of the voltage to suit the sharpness of the part of the electrode at which the denaturation takes place. Optionally, one can conduct the denaturation using a constant current supply rather than a regulated voltage and this may serve to compensate for variations in the geometrical set-up of the electrodes between different denaturation operations.
Where a constant current regime is employed, it will generally be preferable to use a current of from 80 to 160 μA, e.g. about 100 to 125 μA.
As described in WO92/04470, one may employ a promoter compound such as methyl viologen to produce more rapid denaturation. Other promoters are described in WO93/15224, i.e. multivalent cations such as magnesium. Other multivalent cations which are effective and which can be used include lanthanum (La3+). The cations used as the promoters may include inorganic cations complexed with inorganic or organic ligands, e.g. Pt(NH3)6 4+ and Cr(NH3)6 2+.
The promoter may be any inorganic or organic molecule which increases the rate of extent of denaturation of the double helix. It should be soluble in the chosen reaction medium. It preferably does not affect or interfere with DNA or other materials such as enzymes or oligonucleotide probes which may be present in the solution. Alternatively, the promoter may be immobilised to or included in material from which the electrode is constructed.
The promoter may be a water-soluble compound of the bipyridyl series, especially a viologen such as methyl-viologen or a salt thereof. Whilst the mechanism of operation of such promoters is presently not known with certainty, it is believed that the positively charged viologen molecules interact between the negatively charged nucleic acid such as DNA and the negatively charged cathode to reduce electrostatic repulsion therebetween and hence to promote the approach of the DNA to the electrode surface where the electrical field is at its strongest. Accordingly, we prefer to employ as promoters compounds having spaced positively charged centres, e.g. bipolar positively charged compounds. Preferably the spacing between the positively charged centres is similar to that in viologens. Other suitable viologens include ethyl-viologen, isopropyl viologen and benzyl viologen.
Optionally, the process may be conducted using a three electrode system of the kind described in WO92/04470 but generally it is preferred that the volume of solution employed according to this invention is small e.g. 1 ml or less, preferably very small e.g. 100 μl or less, e.g about 25 μl to 40 μl. When using very small reaction volumes of this kind, it will generally not be practical to use a three electrode system.
The process may be carried out at ambient temperatures or if desired at temperatures up to adjacent the pre-melting temperature of the nucleic acid. The process may be carried out at a pH of from 3 to 10, conveniently about 7. Generally, more rapid denaturation is obtained at lower pH. For some purposes therefore a pH somewhat below neutral e.g. about pH 5.5 may be preferred. The nucleic acid may be dissolved in an aqueous solution containing a buffer whose nature and ionic strength are such as not to interfere with the strand separation process.
Preferably, the solution contains a buffer at a concentration at least 10 mM, e.g. about 25 mM. Optionally, the solution may contain further salts such as magnesium chloride and sodium chloride. Preferably, the reaction is conducted in a buffer of the kind used in PCR or in LCR procedures.
Preferably therefore the ionic strength of the solution is above 20 mM, e.g. 25 to 50 mM.
The denaturing process according to the invention may be incorporated as a step in a number of more complex processes, e.g. procedures involving the analysis and/or the amplification of nucleic acid. Some examples of such processes are described below.
We have found that by virtue of the superior cell design described above, it is possible to achieve denatur-ation much more quickly than according to the teachings of WO92/04470 and even in the presence of materials such as PCR buffers which would prevent denaturation or extend denaturation times unreasonably in the apparatus described in WO92/04470. Making use of this shortened time, it is now possible to practice a process of denaturation which is brought to a conclusion within less than 3 minutes e.g. from 1 to 2 minutes or less.
This makes it possible to practice a process of repeated denaturation of double-stranded nucleic acid wherein the nucleic acid is denatured by a process as described above in which the voltage is applied as sequence of repeated pulses having a duration of up to 2 minutes, preferably up to only 1 minute. Between pulses, the voltage may be turned off or reversed for a period which is preferably equal to the period for which the voltage is applied. It is possible to employ pulses of considerably higher frequencies than described above, e.g. from 1 to 100 Hz. Depending upon the purpose for which the denaturation is being conducted, it may not be necessary to achieve any substantial amount of conversion of double-stranded to single-stranded nucleic acid in each denaturation cycle. It may be sufficiently merely to initiate denaturation electro-chemically. For instance, in an amplification procedure, if sufficient denaturation occurs to allow binding of a primer, the extension of the primer by nuclease may be relied upon to displace the unprimed strand of the original nucleic acid from its binding partner over the remainder of the length of the nucleic acid.
Taking advantage of the increased rate of denaturation now obtainable, the invention provides a process of amplifying a target sequence of nucleic acid comprising hybridisation, amplification and denaturation of nucleic acid wherein said denaturation is conducted by subjecting a solution containing said nucleic acid to a voltage applied between electrodes for a period of up to 2 minutes under conditions such as to convert at least a portion of the nucleic acid to a wholly or partially single-stranded form in the solution. Preferably, the electrode configuration used in such a process is as described above. Preferably, the voltage is applied as a repeating pulse having a duration of up to 2 minutes but preferably shorter, e.g. up to 1 minute or even much shorter, e.g. at 1 to 100 Hz.
Preferably, the amplification procedure is PCR or LCR.
Thus the present invention includes a process for replicating a nucleic acid which comprises: separating the strands of a sample double-stranded nucleic acid in solution under the influence of an electrical voltage applied to the solution form an electrode: hybridising the separated strands of the nucleic acid with at least one oligonucleotide primer that hybridises with at least one of the strands of the denatured nucleic acid; synthesising an extension product of the or each primer which is sufficiently complementary to the respective strand of the nucleic acid to hybridise therewith; and separating the or each extension product from the nucleic acid strand with which it is hybridised to obtain the extension product.
In such a polymerase mediated replication procedure, e.g. a polymerase chain reaction procedure, it may not be necessary in all cases to carry out denaturation to the point of producing wholly single-stranded molecules of nucleic acid. It may be sufficient to produce a sufficient local and/or temporary weakening or separation of the double helix in the primer hybridisation site to allow the primer to bind to its target. Once the primer is in position on a first of the target strands, rehybridisation of the target strands in the primer region will be prevented and the other target strand may be progressively displaced by extension of the primer or by further temporary weakening or separation processes.
Preferably, the said amplification process further comprises repeating the procedure defined above cyclicly, e.g. for more than 10 cycles, e.g. up to 20 or 30 cycles. In the amplification process the hybridisation step is preferably carried out using two primers which are complementary to different strands of the nucleic acid.
The denaturation to obtain the extension products as well as the original denaturing of the target nucleic acid is preferably carried out by applying to the solution of the nucleic acid the voltage from the electrodes.
The process may be a standard or classical PCR process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids wherein each nucleic acid consists of two separate complementary strands, of equal or unequal length, which process comprises:
(a) treating the strands with two oligonucleotide primers, for each different specific sequence being applied, under conditions such that for each different sequence being amplified an extension product of each primer is synthesised which is complementary to each nucleic acid strand, wherein said primers are selected so as to be substantially complementary to different strands of each specific sequence such that the extension product synthesised from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer;
(b) separating the primer extension products from the templates on which they were synthesised to produce single-stranded molecules by applying the voltage from the electrode to the reaction mixture; and
(c) treating the single-stranded molecules generated from step (b) with the primers of step (a) under conditions such that a primer extension product is synthesised using each of the single strands produced in step (b) as a template.
Alternatively, the process may be any variant of the classical or standard PCR process, e.g. the so-called "inverted" or "inverse" PCR process or the "anchored" PCR process.
The invention therefore includes an amplification process as described above in which a primer is hybridised to a circular nucleic acid and is extended to form a duplex which is denatured by the denaturing process of the invention, the amplification process optionally being repeated through one or more additional cycles.
The process of the invention is applicable to the ligase chain reaction. Accordingly, the invention includes a process for amplifying a target nucleic acid comprising the steps of:
(a) providing nucleic acid of a sample as single-stranded nucleic acid;
(b) providing in the sample at least four nucleic acid probes, wherein:
i) the first and second said probes are primary probes, and the third and fourth of said probes are secondary nucleic acid probes;
ii) the first probe is a single strand capable of hybridising to a first segment of a primary strand of the target nucleic acid;
iii) the second probe is a single strand capable of hybridising to a second segment of said primary strand of the target nucleic acid;
iv) the 5' end of the first segment of said primary strand of the target is positioned relative to the 3' end of the second segment of said primary strand of the target to enable joining of the 3' end of the first probe to the 5' end of the second probe, when said probes are hybridised to said primary strand of said target nucleic acid;
v) the third probe is capable of hybridising to the first probe; and
iv) the fourth probe is capable of hybridising to the second probe; and
(c) repeatedly or continuously:
i) hybridising said probes with nucleic acid in said sample;
ii) ligating hybridised probes to form reorganised fused probe sequences; and
iii) denaturing DNA in said sample by applying a voltage from an electrode configuration as described to the reaction mixture.
The electrochemical DNA amplification technique can be used analytically to detect and analyse a very small sample of DNA e.g a single copy gene in an animal cell or a single cell of a bacterium.
The temperature at which the process is carried out may be chosen to suit whichever enzyme is used. Thus where Taq is used as polymerase, a temperature of 55 to 68� C. is preferred. If Klenow polymerase is used, ambient temperature will be suitable. It may be desirable to employ known protein stabilisation techniques to avoid electrical damage to the polymerase, especially where a mesophilic polymerase is used.
The invention includes a process for detecting the presence or absence of a predetermined nucleic acid sequence in a sample which comprises: denaturing a sample double-stranded nucleic acid by means of a voltage applied to the sample in a solution by means of electrodes spaced as described above; hybridising the denatured nucleic acid with an oligonucleotide probe for the sequence; and determining whether the said hybridisation has occurred.
Thus, the invented process has application in DNA and RNA hybridisation where a specific gene sequence is to be identified e.g. specific to a particular organism or specific to a particular hereditary disease of which sickle cell anaemia is an example. To detect a specific sequence it is first necessary to prepare a sample of DNA, preferably of purified DNA, means for which are known, which is in native double-stranded form. It is then necessary to convert the double-stranded DNA to single-stranded form before a hybridisation step with a labelled nucleotide probe which has a complementary sequence to the DNA sample can take place. The denaturation process of the invention can be used for this purpose in a preferred manner by carrying out the following steps:
denaturing a sample of DNA by applying a voltage by means of an electrode configuration as described to the sample DNA with optionally a promoter in solution or bound to or part of the structure of the electrode;
hybridising the denatured DNA with a directly labelled is or indirectly labelled nucleotide probe complementary to the sequence of interest; and
determining whether the hybridisation has occurred, which determination may be by detecting the presence of the probe, the probe being directly radio-labelled, fluorescent labelled, chemiluminescent labelled or enzyme-labelled or being an indirectly labelled probe which carries biotin for example to which a labelled avidin or avidin type molecule can be bound later.
In a typical DNA probe assay it is customary to immobilise the sample DNA to a membrane surface which may be composed of neutral or charged nylon or nitrocellulose. The immobilisation is achieved by charge interactions or by baking the membrane containing DNA in an oven. The sample DNA can be heated to high temperature to ensure conversion to single-stranded form before binding to the membrane or it can be treated with alkali once on the membrane to ensure conversion to the single-stranded form. The disadvantages of such methods are:
heating to high temperature to create single-stranded DNA can cause damage to the.sample DNA itself.
the use of alkali requires an additional step of neutralisation before hybridisation with the labelled probe can take place.
One improved method for carrying out DNA probe hybridisation assays is the so-called "sandwich" technique where a specific oligonucleotide is immobilised on a surface. The surface having the specific oligonucleotide thereon is then hybridised with a solution containing the target DNA in a single-stranded form, after which a second labelled oligonucleotide is then added which also hybridises to the target DNA. The surface is then washed to remove unbound labelled oligonucleotide, after which any label which has become bound to target DNA on the surface can be detected later.
This procedure can be simplified by using the denaturing process of the invention to denature the double-stranded DNA into the required single-stranded DNA. The working electrode, counter-electrode and optionally a reference electrode and/or a promoter can be incorporated into a test surface or a well in which the DNA probe assay is to be carried out. The DNA sample and oligonucleotide probes can then be added and the voltage applied to denature the DNA. The resulting single-stranded DNA is hybridised with the specific oligonucleotide immobilised on the surface after which the remaining stages of a sandwich assay are carried out. All the above steps can take place without a need for high temperatures or addition of alkali reagents as in the conventional process.
The invention includes an electrochemical cell for use in the denaturation of nucleic acid comprising a well having an electrically conductive inner surface constituting a first electrode, and a second electrode dipping into said well and reaching to close proximity to the floor of said well. Preferably, the well is formed in a body of conductive material constituting the second electrode. Both the well and the second electrode may be of conductive carbon such as graphite.
The invention further includes a kit for use in a nucleic acid assay or amplification comprising an electro-chemical cell of this kind and at least one of a nucleic acid for denaturation, an oligonucleic acid, one or more nucleotides, a polymerase, a ligase and/or a buffer solution.
The invention will be further described and illustrated with reference to the accompanying drawing which is a cross-sectional view through a cell for use in accordance with the invention.
The cell illustrated comprises a graphite block 10, containing a 4 mm diameter well 12 on its upper surface, constituting a first electrode. A second electrode formed from a 2 mm diameter graphite rod 14 having a tapering end portion 16 is pressed into the well through an insulating collar of plastics material 18. The rod is adjusted down-wards in the well until it forms a short circuit and is then lifted back by as little as possible to open the circuit again. The capacity for liquid for the cell is approximately 25 μl. A DC voltage is applied to the apparatus with the well being made positive with respect to the rod by preferably 1.6 to 2.4 volts.
Although the rod electrode shown in FIG. 1 is blunt ended, it provides a sharp edge between its flat end and its tapering frustoconical surface. An optional alternative configuration is for the rod to be sharpened to a point. This may be achieved by using a conventional pencil sharpener. The resulting electrode may be further smoothed using a blade or abrasive. Using such a pointed electrode, the capacity for liquid of the well is increased to 40 μl.
Multiple processes according to the invention may be carried out simultaneously in apparatus containing a multiplicity of sample receiving wells each provided with a respective electrode pair, one electrode in each case optionally being the well itself. In a preferred form for such apparatus a block containing the wells has a liftable lid with electrodes depending therefrom into the wells. Each well may have a pair of electrodes on the lid in various possible electrode conformations such as parallel rods, parallel plates, optionally of mesh, or coaxial hollow cylinders, again optionally of mesh. Alternatively, single electrodes may be provided on the lid for each well and the block containing the wells may be conductive and may serve as a common second electrode. The block of wells may also contain respective electrodes for each well.
The lid may comprise a flat plate portion bearing the electrode or electrodes for each well and a separate backing member bearing electrical connections and circuitry which connects up the electrodes when the two parts are assembled. A single electrical supply to the unit may be split by said circuitry and supplied in a controlled manner to the electrodes such that each electrode is controlled, e.g. to a constant voltage or constant current. The plate portion carrying the electrode array may thereby be replaceable without replacement of the control circuitry and may be made disposable. The plate portion and the backing member may be aligned with one another on assembly by locating pins and apertures and may similarly by aligned with the block containing the wells, which also may be disposable.
The following examples illustrate the use of the apparatus described above and methods according to the invention in comparison to techniques described in WO92/04470.
Denaturation of DNA was carried out in the cell shown in FIG. 1, modified by the use of a pointed electrode 14, using a constant voltage of 2.4 V. Denaturation of 40 μl of calf thymus DNA (12 μg/ml) in 1 mM methyl viologen was completed in 1 to 2 minutes.
Using the three electrode electrical system described in WO92/04470, DNA was denatured using -IV at the working electrode with respect to the calomel reference electrode (�2.4V). Denaturation of 1 ml of calf thymus DNA (4 μg/ml) in 1 mM methyl viologen was completed in about 25 minutes.
Denaturation of calf thymus DNA was carried out using a constant voltage of 2.4 V between the electrode of the cell of FIG. 1, modified by the use of a pointed electrode 14. Denaturation of 40 μl DNA (20 μg/ml) in 25 mM KPO4, pH 6.0, 1 mM methyl viologen was completed in 1 to 2 minutes.
Using the three electrodes of WO92/04470 DNA was denatured using -IV at the working electrode with respect to the calomel reference electrode (�2.4V). Denaturation of 1 ml calf thymus DNA (4 μg/ml) in 25 mM Tris, pH 7.5, 1 mM methyl viologen, required more than 180 minutes.
Whilst the invention has been described above in terms of the preferred embodiments illustrated by the examples, many modifications and variations thereof are possible within the scope of the invention.
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Hiv Workplace Rules
Here's a taste of what TheBody.com has to offer on this topic:
Get the Facts
HIV and the Brain...disorder. Additional Reading: Lapp, D. C. (2004). Don't forget!: easy exercises for a better memory at any age . US: Barnes and Noble Books. ( Buy it online .) Lapp, D. C. (1998). Maximizing your memory power: memory in the workplace . Hauppauge, NY: Barron's Educational Series, Inc. ( Buy it online .)
Ask the Experts
Can I work in the hosp. if i'm positive?Just passed Nclex-RN and has been offered a job in PCU at a local hosp. Will they still hire me after the test comes back positive?
Response from Nancy Breuer
WorkPositive, Inc....Hospitals all set their own rules on potential exposure to HIV in the workplace. Usually they do not restrict the activities of someone who's not doing invasive procedures. Ask for a copy of your hospital's Read more »
Becoming a workplace educatorHow can I learn more about becoming an HIV/AIDS workplace educator?
Response from Nancy Breuer
WorkPositive, Inc....Responds to AIDS Program, and WorkPositive, my own company (www.workpositive.com). However, there's about as much demand for workplace HIV educators right now as there is for electric typewriter repairpeople, despite the still-lamentable level of... Read more »
Mandatory HIV Education in the Workplace?I work at a private university in a relatively rural area. HIV education does not take priority even though we are training medical professionals. I am a volunteer for several local HIV/AIDS agencies, and am of the opinion that HIV education...
Response from Lynn L. Franzoi
Worldwide Benefits for Fox, Inc.No. Read more »
Thank you and research on HIV/AIDS in workplace continuesThank you for your response about NGO's HIV/AIDS workplace policies. I will definitely check out the website that you suggested. I will be glad to share the results of my research when I finish. I noticed in your bibliography that you worked with...
Response from Nancy Breuer
WorkPositive, Inc....there are chapters. From the questions I've received, it would appear that CPR instructors are not required to have training in HIV from any angle, but I may be dealing with the exceptions rather than the rule. At ARC's national headquarters in... Read more »
Kenyan Government Developing Policy to Address HIV/AIDS in Workplace, Official Says... The Kenyan government plans to develop a policy to address HIV/AIDS in the workplace, Mark Bor, secretary of the Ministry of Labour and Human Resource Development , said on Sunday at the opening of a two-day seminar Read more »
Zidovudine (Retrovir, AZT)...benefit to taking zidovudine. But zidovudine is used as part of combination therapy for people who are exposed to HIV through a workplace accident (needlestick or splash). Zidovudine greatly reduces transmission of HIV from the mother to her... Read more »
Women and HIV/AIDS... Table of Contents A Look at the Numbers Is HIV Different for Men and Women? Treatment in HIV+ Women: Efficacy, Side Effects, and Drug Interactions Gynecological Issues Read more »
How to Spot HIV/AIDS Fraud... What Is HIV/AIDS Fraud? Warning Signs of an HIV Fraud Common Sense About Unproven Treatments Questions You Should Ask Take Your Read more »
Connect With Others
Posted by toronto111, 2 Replies...I've been known to have very bad coughs like that before in wintertime, especially in enclosed dusty areas like my home and my workplace, and I've been under a fair amount of stress as I've been recovering from my fairly deep depression. I'd like... Read more »
Workplace issues and HIV/AIDS
Posted by ScotCharles...http://www.thebody.com/workplace.html Check out this link for workplace issues with HIV/AIDS. It is very thorough. Read more »
William is in the hospital - Please Pray for Him!
Posted by Survivor, 16 Replies...half way across the world and his surgeon is well respected. Ben does not think the mass could be anyway related to his hiv since he is got such great numbers 770+ and vl of 1700. I just hope and pray that whatever this is, its benign. ... Read more »
window period
Posted by Anonymous, 5 Replies...Mixed-HIV-Status Couples Strategies for Treatment- Experienced HIVers Tratamientos (En Espaol) Understanding Your Labs Women and HIV Workplace and Insurance Issues AIDS-Related Cancers Lipodystrophy and Wasting Nutrition and Exercise ... Read more »
Resource Centers
Guides for People With HIV


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'Mad Men' Season 6 Primer: Where the Show Left Don Draper, the Agency and the '60s
Matthew Weiner's beloved yarn of admen, American history and infidelity returns to AMC on April 7 with its hefty ensemble likely still dealing with the events of the fifth season.
Mad Men can be hard enough to keep track of without the nine-month lapse in seasons. Characters drift in and out, the styles of the '60s evolve, and one episode can suddenly find one of the gorgeous stars suffocating in a fat suit.
So for those tuning into the AMC drama's season-six opener on Sunday, April 7, now is probably a good time for a quick refresher in where we last saw Don Draper and his many cohorts.
Don Draper (Jon Hamm): Dealing with a toothache and remorse for the death of Lane Pryce, Don finally caved to his new wife's pleas to assist her acting career. He was last seen alone at a bar being approached by a woman. "Are you alone?" she asked. Cue ambiguous Draper face and a signature Mad Men cliffhanger.
PHOTOS: 'Mad Men' Season 6 Premiere
Peggy Olson (Elisabeth Moss): The squishy center of the jelly doughnut that is Mad Men, Don and Peggy's odd and indelible bond, seemed to be in jeopardy when Peggy abruptly left Sterling Cooper Draper Pryce for a pay raise and title improvement at CGC. But the two reconnected during a chance encounter at a movie theater, and a still-empowered Peggy finished the season happily coupled with boyfriend Abe and gawking at humping dogs outside of her Richmond motel room.
Roger Sterling (John Slattery): Our last glimpse of Roger was technically of his butt, as the single-yet-again playboy looked out at the city from his hotel room, discovering himself with another acid trip. LCD had previously prompted the existentially stalled adman to dump wife Jane, but he seemed at peace with his decision.
Betty Francis (January Jones): After the shock of Betty's prosthetics and fat suit wore off, everyone's favorite morally bankrupt mother seemed to lighten up a bit. She and Sally ended the season on a rare moment of connection, though husband Henry Francis still seems more or less incidental. And her mother-in-law would drive anyone to binge eating.
Pete Campbell (Vincent Kartheiser): Though Mad Men has hinted at Pete's tenuous mortality on more than a few occasions, it really dangled the snide account executive over the proverbial pit when he spectacularly failed at adjusting to married life in the suburbs. His affair with Beth (Alexis Bledel) drove her back to the arms of electroshock therapy, which caused her to forget he ever existed. Broken, Pete spilled the beans about the tryst to Beth's husband, who left him bloodied and bruised -- but still alive!
Sally Draper (Kiernan Shipka): During an especially ill-advised jaunt to the Natural History Museum with creepy Glenn, Sally got her period. It was an awkward end to a season that saw the most adorable Draper dress up and go to the Codfish Ball as Roger's date, but it did seem to put her back on good terms with Fat Betty.
Joan Harris (Christina Hendricks): Ignoring, for a moment, the characters who didn't make it through the last season, Joan might have had the toughest road of late. Things went south for the new mother when her husband, Greg the rapist doctor, returned home from Vietnam. He voluntarily reinlisted, she threw him out, and he filed for divorce. Joan took another hit when she slept with a Jaguar executive to secure his account. On the bright side, she made partner and finally seemed to officially segue into the boys' club.
Megan Draper (Jessica Pare): After singing her way into our hearts, poor Megan saw a lot of the joy sapped from her during season five, realizing that being Mrs. Draper isn't the easiest title to have and the advertising game just wasn't her shtick. After she bowed out of her job at Sterling Cooper Draper Pryce, she took acting classes, appeared in an awkward play and finally convinced Don to help her get a job in a commercial.
Lane Pryce (Jared Harris): Uh oh, that's right. The title partner of Sterling Cooper Draper Pryce is now that in name only. Driven to a mental break by mounting debt, the Brit hung himself in his office toward the end of season five.
The 60s: Covering just about a year, the most recent season of Mad Men left off in spring of 1967. The Civil Rights movement was reaching its crescendo, opposition to the Vietnam War was mounting, and Lyndon B. Johnson was serving his final term as President of the United States. If you're placing bets on historical subplots for this year, odds are in favor of the 1968 presidential election, the Chicago DNC protests and the assassinations of Robert F. Kennedy and Martin Luther King Jr.
Email: email@example.com; Twitter: @MikeyLikesTV
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Michelle Monaghan Battles Motherhood and Afghanistan in Fort Bliss
Ask and ye shall receive: after we wished True Detective's Michelle Monaghan would stop getting underwritten supporting roles, a trailer for her new movie, Fort Bliss, is out. »
Ask and ye shall receive: after we wished True Detective's Michelle Monaghan would stop getting underwritten supporting roles, a trailer for her new movie, Fort Bliss, is out. » 4/08/14 4:40pm 4/08/14 4:40pm
I love True Detective. I love the eerie, deserted bayou setting, Woody Harrelson and Matthew McConaughey playing against type as well as playing off each other, and the themes of existential horror as personified by that hulking figure in the tighty whities and the gas mask. » 3/03/14 6:00pm 3/03/14 6:00pm
In an interview with the magazine Glam Belleza Latina, Salma Hayek managed to make the 50 pounds she put on when she was pregnant with daughter Valentina in 2007 sound like she spent 6 months in a cage playing mah jongg with the pinheads in the 1932 movie Freaks. » 6/15/13 11:30am 6/15/13 11:30am
How do stars know which events are compulsory? Like, how do they hear about an "Evening of Cocktails and Shopping to Benefit the Children's Defense Fund" and know everyone will be there, looking fabulous? Maybe there's some secret cabal of mind-melding managers... » 4/21/11 10:30am 4/21/11 10:30amRosario Dawson makes simple neutrals stunning. …
You've seen the Source Code poster, in which Jake Gyllenhaal has to strike all kinds of uncomfy action poses. Luckily, the premiere was more restrained. Some of it, anyway. » 3/29/11 10:43am 3/29/11 10:43amThere are so many things to look at here that even the irrepressible Vera Farmiga is virtually hidden. Sometimes Jake Gyllenhaal just looks…
The world premiere of Iron Man 2, at Hollywood's El Capitan Theatre, was so chock-a-block with strange shoes and sexyface and shiny, unhappy dresses and short-shorts that we're as exhausted as if we'd been clomping around in an iron suit. » 4/27/10 10:30am 4/27/10 10:30am
The Calvin Klein Collection & Los Angeles Nomadic Division 1st Annual Celebration For L.A. Arts Monthly and Art Los Angeles Contemporary - yes that's the name - was star-studded and typically understated...except when it wasn't. Like when Jared Leto appeared. » 1/29/10 10:30am 1/29/10 10:30am
It's going to be okay. After the train-wreck that was the Golden Globes, the stars all showed, chastened and sheepish and back in form for Saturday's SAGs. Christina, Marion, Penelope...it's so nice to have you back! » 1/25/10 10:20am 1/25/10 10:20am
The Cinema Society & Hugo Boss screening of Inglourious Basterds in NYC was star-studded, full of win, and had a gratifying dollop of awful. In short, it's a good one, kids. And supersized! » 8/18/09 10:30am 8/18/09 10:30am


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Oh go crawl back in your hole.
Oh go crawl back in your hole. It's been posted many times already.
10th Amendment - The powers not delegated to the United States by the Constitution, nor prohibited by it to the States, are reserved to the States respectively, or to the people.
There is no power that was delegated to the federal government to make legal tender. Legal tender does not mean "coin money" or "emit credit", and "make" here is used in the legal sense of enforce.
All the terms have been discussed many times already. You aren't actually ignorant. You're just a liar - and one quick to accuse others of it when they point out why your posts aren't matching reality.
And for the support of this Declaration, with a firm reliance on the protection of Divine Providence, we mutually pledge to each other our lives, our fortunes and our sacred honor.
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Related Publications
- Related by Author
- Related by Topic
The Uncertain Future of American Jewry-Israel Relations
Middle East Institute (Jerusalem), Summer 1984
Topic: Political Behavior, Diaspora Relations, Israel-Diaspora Relations, International Relations, Politics, US-Israel Relations
Name of Publication: Jerusalem Quarterly
Volume/Issue: No.32
Genre: Scholarly Journal
Coverage: Israel , United States
Language: English
Copyright Information: Download for personal use, freely distribute link
Bibliographic Information:
Sheffer, Gabriel. The Uncertain Future of American Jewry-Israel Relations. Jerusalem Quarterly. Middle East Institute (Jerusalem). Summer 1984: http://www.bjpa.org/Publications/details.cfm?PublicationID=4410
» View Publication
(PDF, 1.24 Mb)


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Dr. Chad D. Ringley, MD
- 5415 Fashion Square Blvd
Saginaw, MI 48604
- 2 Locations
- (989) 497-9667
Dr. Chad D. Ringley graduated from the Indiana University School of Medicine in 1998. He works in Saginaw, MI and 1 other location and specializes in Surgery and Bariatrics. Dr. Ringley is affiliated with Covenant Healthcare, Covenant Medical Center Rehabilitation, Mercy Health Saint Marys and Saint Marys Of Michigan Medical Center.
Gender: Male
Languages Spoken: English
Surgery, Bariatrics
Medical School: Indiana University School of Medicine; Graduated 1998
*This physician has multiple office locations. See Office Information for details.


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OSI-Approved Open Source (140)
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Broadway darling Kelli O'Hara invited Broadway.com inside her dressing room at Nice Work If You Can Get It to show off her five favorite things, including photos, notes and her grandfather's flask.
Broadway darling Kelli O'Hara invited Broadway.com inside her dressing room at Nice Work If You Can Get It to show off her five favorite things, including photos, notes and her grandfather's flask.September 12, 2012
The charismatic actor shares five must-have dressing room items.November 16, 2011
When he's not scheming onstage as Black Stache in Peter and the Starcatcher, Matthew Saldivar goofs off inside his dressing room at the Brooks Atkinson Theatre, where he proudly shows off his ukulele and at least one pair of smelting glasses.September 19, 2012
When Madison Dirks isn't slinging back bourbon in Who's Afraid of Virginia Woolf?, he's relaxing in his dressing room at the Booth Theatre. Check out his favorite things, including Louisiana swag, an alcoholic ornament and more.February 14, 2013
The Tony-nommed actor reveals the most important items in his dressing room.May 23, 2012
The leading man reveals five favorite dressing room items.January 4, 2012
The actress shares her five favorite dressing room items.January 11, 2012
The actor shows off five favorite items backstage at the Shubert Theatre.February 8, 2012
Margaret Colin, who plays John Lithgow's buttoned-up wife in The Columnist, breaks loose when she steps offstage! Colin shows off the dried flowers, grits and liquor-laced Kombucha that make her dressing room to die for!June 13, 2012
The actress shares five of her favorite dressing room items.February 14, 2012
It seems like Jeremy Kushnier has sticky fingers! From Andy Karl's guitar to Jesus Christ's shake weight, the Jersey Boys star shows off some of his favorite backstage items, few of which are actually his!November 7, 2012
The mama of Memphis shows off her backstage essentials.September 1, 2011


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Whether you have a logo, need one tweaked or designed from scratch (browse logo portfolio), we can build upon it to create a consistent, graphically appealing identity package that's uniquely yours … setting the stage for all subsequent marketing and sales materials.
Letterhead, business card, envelope pocket folder and notepad
See more of this client's work:
Print design
Direct marketing
I'm really impressed with what you put together and you both have been a pleasure to work with. I'm looking forward to working with you more as we grow. The feedback on the site has been tremendous!
Jennifer Remling
Carve Your Own Road


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Mothers' Day Event
- Performing & Visual Arts
- Amway Center, Orlando FL
Event Tickets
Corporate Event Tickets
Saturday, July 26, 2014 from 7:00 PM to 10:00 PM (EDT)
NewAvenue Blast Talent Show is a 3-hour talent show event to discover new talents. Register and buy your tickets online. Ther will be surprises. Tickets bought 2 weeks in advance will also receive one of my books to structure your business.
NewAvenue is a private company that sells software, markets, and promotes businesses. The NewAvenue has three main products that include: DealerPros, Eve NewDirection, and TheNet. The NewAvenue offers various ways to earn an extra income. Ticket: College students be a TheNet member and get your ticket
Be a winner and win the prize
Be a Promoter and earn %10 on your investment
Be a TheNet Member and earn %17 per Tickets Activity Shares
To learn more visit https://www.newavenuellc.org
In order to purchase these tickets in installments, you'll need an Eventbrite account. Log in or sign up for a free account to continue.


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The Top 10 In Action Sports
01Welcome to Qasr Al-Farid: The Rock With A Door That Will Rock Your World
0218 Things That Will Give Anyone Who Did Duke Of Edinburgh A Serious Flashback
0316 Things That Will Give 90s Skater Kids A Serious Flashback
04This 10 Year Old Kid Just Nailed A Dub Cork 1080... On His Second Attempt
05The 10 Most Spectacular Road Trips on Earth
06Water Slides, Plane Wings and Danny MacAskill: This is Road Cycling Like Never Before...
0724 Reasons Why Snowboarding Was Better In The Early Noughties
08Could Scientists Be About to Build a Terminator? They're Closer Than You Think…
09Win a Holiday for 2 in Chamonix with Three #holidayspam
1017 Holiday Beer Photos That Every Skier Takes to Make Their Friends Jealous
The Eagle Has Landed
A Surfing video by talon1palmMEDIA - 5,754 views
Shot by onepalmMEDIA.com on Thursday 15th & 16th March using our Red Epic shot 2K @ 240fps with custom Dave Kelly housing ( email@example.com ) using 5.7mm Kinoptik and 40mm Zeiss Distagon Mk2 lenses. The day after the 2013 Quiksilver Pro finished, Kirra turned on in a way that it hadn't done so for the last 10 - 15 years. Clear skies, clear water and a nice groundswell courtesy of Tropical Cyclone Sandra pulsing endless lines into the once fabled Gold Coast point break of the 70's, 80's and 90's. I admit it, I blew it.... I got there late but decided to swim out and shoot from the water. Would've loved to have been there when all the best surfers in the world were ruling it, but I was walking on the beach at Broadbeach with my 5 month old son. It is what it is, C'est La Vie, etc...... This clip, it's not gonna be everyone's cup of java..... cos it's not 2 minutes long with the latest on trend garage band, but I do hope you enjoy the art in it and fall in love with the ocean like I do every time I envelop myself in it. I'd like to thank Mind Tree ( soundcloud.com/mindtree ) for the tunes and Red Digital Cinema ( red.com ) for making an unreal piece of equipment that I am constantly impressed with. Check out onepalmMEDIA.com for other clips on my website that hasn't been updated since it was originally posted. Hahahaha.... anyone wanna be my web guy? Must know wordpress. Email me at - email@example.com Aloha OPM


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LSU's Miles pressing players to clean up mistakes
Updated: October 2, 2012, 5:06 AM ETAssociated Press
BATON ROUGE, La. -- Lackluster efforts in victories against Auburn and Towson have fourth-ranked LSU needing to make an adjustment as it begins the heart of its Southeastern Conference schedule this week.
The Tigers will take on 10th-ranked Florida at "the Swamp" in Gainesville, Fla., on Saturday.
Following the game against the Gators, LSU will meet three more currently ranked opponents in the next four games -- No. 6 South Carolina, No. 1 Alabama and No. 20 Mississippi State.
Tigers ...
Read full story
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By: erikduda (59) in BU Today
YouSpeak asks, "Do people use their cell phones too much?"
bu, boston, university, youspeak, cell phone, usage, question, overusing, overuse
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Jobs Companies & Reviews Companies Salaries Interviews
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"I should have been prepared for this one, but apparently it was still a surprise. What was my biggest challenge in the previous place of employment and how did I bring it to succesfull resolution."
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Listen to the Ben Shapiro Show on AM 770 KTTH (weekdays from 3-6 p.m.) from May 12th – 23rd for your chance to win a pair of tickets to see In My Life – A Musical Theatre Tribute to the Beatles!
In My Life – A Musical Theatre Tribute to the Beatles is the award-winning musical biography of the Beatles through the eyes of manager Brian Epstein, featuring the live music of renowned tribute band, Abbey Road. The show is widely considered by industry insiders to be the most unique Beatles show in decades.
More than just a Beatles tribute concert, In My Life gives the audience a chance to "be there" at pivotal moments in the extraordinary career of the Beatles – from Liverpool's legendary Cavern Club, to the Ed Sullivan Show, Shea Stadium's 50,000 + screaming fans and their final live performance on the rooftop of their Apple Corp offices. With manager Brian Epstein serving as Narrator, In My Life allows the audience to get a glimpse inside the world of the Beatles from their point of view, as well as hear some of the greatest songs ever written.
The show is appearing at the following Pacific Northwest locations from June 3 – 8:
- Bremerton: Tuesday, June 3rd at the Admiral Theatre. Tickets are $25 – $55 and may be purchase online at admiraltheatre.org.
- Edmonds: Wednesday, June 4th at the Edmonds Center for the Arts. Tickets are $35 – $55 and may be purchased online at edmondscenterforthearts.org.
- Bellingham: Thursday, June 5th at Mount Baker Theatre. Tickets are $20.99 – $39.99 and may be purchased at mountbakertheatre.com.
- Portland: Friday, June 6th at the Newmark Theatre. Tickets are $40 – $60 and may be purchased online at ticketswest.com.
- Longview: Sunday, June 8th at the Columbia Theatre. Tickets are $25 – $40 and may be purchased online at columbiatheatre.com.
The show is appropriate for all ages.
Top stories
David Boze: Seattle mayor is reading Indiana law all wrongApril 1, 2015David Boze says Seattle Mayor Ed Murray hasn't looked at what the Indiana discrimination law actually says.
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It's more complicated than thisMarch 18, 2015David Boze says smearing the wage gap as primarily or even significantly a product of discrimination is political manipulation
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Economic policies of the left are killing middle class in King CountyMarch 13, 2015Shapiro: If you're a middle class person, the reason you can't live in King County is because it's just too expensive.
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Ospreys players banned from getting fake tans
- 3 November 2011
- From the section Front Page
Welsh rugby union side Ospreys has banned its players from getting fake tans.
The players have also been told they cannot wear coloured boots until they have played 50 times for the side, or racked up 15 international caps.
"The fake tan and the coloured boots and the Galacticos label doesn't interest us," coach Sean Holley told the Daily Telegraph newspaper.
Former Ospreys' player Gavin Henson is known for his spray tan.
Banning the tan is an attempt to build a new identity for the club after it recently lost key players, like Welsh international James Hook.
"It's real tan only," said Holley, who added that the right to wear coloured boots had to be "earned" by playing regularly for club or country.
"We want to work hard, we want to represent our region properly", he said.
"We don't want to be false or misrepresented."
Ospreys' new approach is showing signs of paying off - the team is currently topping the Pro12 table after winning six of their opening seven games.
"We are trying to get good people, good young lads through who represent us well and I think we are achieving that at the start of the season," said the coach.


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Known as the "Emperor," Shields Green played a role in John Brown's Raid on Harper's Ferry. As a young man he escaped through the Underground Railroad network to Rochester, New York, where Frederick Douglass provided shelter and introduced him to radical abolitionists. He accompanied Frederick Douglass to Chambersburg, Pennsylvania, where the great abolitionist spoke to John Brown for the last time. Brown was unsuccessful in convincing Douglass to join him in the raid; he did, however, recruit the young Green.
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This hotel is right on the airport grounds with a walkway to terminal. It was quiet; clearly the weekends are not when they are busiest. The room was contemporary in decor, large, and had a great bathroom. There are a number of restaurants, not all of which are open on the weekend. We had a nice late afternoon lunch in one of the restaurants/bar, and it was perfect German comfort food fare for a cold, rainy afternoon. We also were able to take two short train rides relatively easily to the old town area of Dusseldorf.
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Black plain weave slim fit 1 button jacket
Item No.46497_0035463A
The Ben Sherman tailoring collection features the clean lines and finishes that are drawn from the brand's unrivalled British heritage and mod roots. The range has three fits - the regular City fit, the slim Kings fit and the short and skinny Camden fit.
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- Slim - Cut closer to the body. Perfect for slimmer frame
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Please join us for these exciting events!
Tuesday, March 31, 2015, 11: 30 a.m., Olin Library, Room 142 "Archiving the Myth That Shot Liberty Valance," a talk by Stevphen Shukaitis, Senior Lecturer, University of Essex
Shukaitis is a recipient of a travel grant from the Department of Special Collections. In his talk, Sukaitis will raise the question of how to relate to and archive materials produced by artists and cultural figures that have a dimension of myth-making in them. Such artists may intertwine historically accurate materials and invented or fabricated materials in ways that make it hard to tell what is "real" and what is invented. Attendees are welcome to bring their lunch.
Tuesday, March 31, 2015, 5:30-7:30 p.m., Olin Library, Ginkgo Reading Room. Panel Discussion about Art Exhibit: A.I.R. Refreshed
A mixed media traveling exhibition called A.I. R. Refreshed is displayed in Olin Library's Ginkgo Reading Room and the Grand Staircase Lobby through April 17. It presents contemporary artwork from A.I.R. Gallery in New York. Founded in 1972, that gallery was the first non-profit cooperative dedicated to showing work by female artists. Kat Griefen, former director of A.I.R. Gallery, will present remarks, after which a panel moderated by Elizabeth Childs, professor of art history, will respond. Panelists are Heather Bennett, artist and Sam Fox faculty member; Helen Kornblum, an art collector specializing in art made by women; and Yvonne Osei, a fellow in the Graduate School at Sam Fox. A reception follows.
Co-sponsors: Women, Gender, and Sexuality Studies Program; Department of Art History & Archeology; Mildred Lane Kemper Museum; Graduate School of Art; Sam Fox School; Law Culture and Identity Initiative; Center for the Humnities; and University Libraries.
Thursday, April 2, 2015, 4 p.m., Olin Library, Ginkgo Reading Room. "Diderot Project: Research & Artifact" talk by Ken Botnick
Professor Ken Botnick will discuss the evolution of his recently completed limited edition artist's book, a visual and textual meditation on concepts encountered in DIderot's Encyclopédie, a holding of the Libraries' Department of Special Collections. Botnick will speak about concepts of the hand and craft, tools and machines, labor and leisure, memory, sensation and perception. Books will be on display in Special Collections. Introduction by Professor Heather Corcoran. A reception follows.
Wednesday, April 8, 2015, 5 p.m., Umrath Lounge. "Fire in the Library" : a lecture by Sarah Thomas, Vice President of Harvard Library
Librarians are the keepers of the flame of culture, history, and knowledge, and their mission is to light the way to discovery. Harvard Librarian Sarah Thomas explores the transformation of libraries in the 21st century, even as they preserve enduring core values. A reception follows.
Sarah Thomas, Ph.D. has led Harvard Library since 2013. From 2007 to 2013, she was the Bodley Librarian at the University of Oxford, the first American and the first woman to do so. For the decade prior to that, she was University Librarian at Cornell University, and earlier held leadership positions at the Library of Congress, the National Agricultural Library, the University of Georgia, Stanford's Research Libraries, Johns Hopkins, and Harvard.
This is the Jack E. & Debbie T. Thomas Endowed Lecture, presented in honor of retired Dean of Libraries Shirley K. Baker.
If you plan to attend the Sarah Thomas lecture please email or call Joy Lowery at email@example.com or 935-5418 with your name and the number of persons in your party. An RSVP is not required but will help us in planning the event.
For questions, contact Brian (firstname.lastname@example.com ; 5-4824 ; IM/chat ; Olin, rm. 124)


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Experimental off dock drop yard at the Port of Los Angeles could help speed containers.
Businesses large and small and across many sectors share their views on the massive West Coast port cargo delays.
Disney and Dollar General described their strategies to avoid West Coast port congestion mess at the TPM conference.
The German ocean carrier said its financial results were impacted by costs from merger with Chilean liner CSAV.
ConAgra and Lululemon said they expect shipment delays for several more months as a result of West Coast port congestion.
The new container service will connect ports in China, Taiwan and South Korea with Mexico, Colombia, Peru and Chile.


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I live in the city, I have children who are a part of my life. I am looking for connection and to spend time with a woman who is not afraid to ride the subway, walk through the city, see the cultural side as well as the other side. Who will accept what life has to offer, and see the positive in things. I enjoy seeing live music, just as comfortable at the art museum as walking in the wissahickon. I could write a bunch bs here to try and look good and that's all it would be is bs. So, cheers, happy new year.
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Even though Arizona's population has remained stagnant since the onset of the economic recession, Gary Pierce says members of the Arizona Corporation Commission should be planning as far as half a century into the future to be ready for the growth that will happen.Read More »
Tag Archives: energyFeed Subscription
David Bradley: Commission needs to act as 'catalyst' to create jobs
Beyond merely serving as an elected member of a regulatory agency, David Bradley says he regards having a seat on the Arizona Corporation Commission as having the opportunity to be an agent for big change.Read More »
Brenda Burns: Commission should work better with Legislature on solar
In seeking a seat on the Arizona Corporation Commission over Democratic rivals, Scottsdale Republican Brenda Burns is touting her private-sector experience, going back 30 years when she and her former husband ran an optometry business.Read More »
Jorge Luis Garcia: Keep Legislature away from Commission's solar power authority
Jorge Luis Garcia says he's bit worried that he is a Tucson state senator who has little name recognition in the Phoenix area, where most of the state's votes are found.Read More »
Arizona firm banks on farm troughs for commercializing algae
Arizona, with its sunny, warm climate has been hailed as the ideal region for algae biomass production. Proponents hope that algae, which can contain up to 60 percent oil, can some day squeeze out a petroleum substitute that could be produced at a price low enough to compete with natural gas.Read More »
Arizona stimulus funds largely unspent
Many deadlines to use stimulus money fall in 2012. That means the next 18 months offer some guaranteed work for construction crews, lower energy bills for some homeowners and perhaps even a benefit for taxpayers as government buildings become more efficient.Read More »
Leading renewable energy manufacturer opening plant in Phoenix, adding 350 jobs
Power One Inc., the nation's largest manufacturer of renewable energy conversion devices, announced Thursday that it will open a manufacturing facility in Phoenix.Read More »
State's energy future at stake in Corp. Comm. election
The next Arizona Corporation Commission will determine policies that impact the daily lives of Arizonans perhaps more than most well-known elected offices in the state. Established by the Arizona Constitution, the commission regulates public utilities, corporate filings and securities, and railroad and pipeline safety.Read More »
Soaring over solar?
In Arizona, power generated by the sun is the darling of many renewable-energy advocates, solar-power based companies and the governor. There is, however, another less cost-intensive player trying to gain a foothold in the renewable power game: wind.Read More »
When the slop is in the trough
When the slop is the trough all the pigs will be there. And many were there recently at Tom's Restaurant & Tavern for a panel discussion on renewable energy standards, specifically the 15 percent mandate passed by the Arizona Corporation Commission. Actually, the discussion was more about government subsidies than renewable energy standards; especially with respect to the notion that "everyone knows" these subsidies are needed to promote Arizona's nascent solar industry.Read More »


text:
Pronunciation: coercizione
Added on: 2010-08-06 Listened: 13 timesin: sostantivo
Pronunciation in Italian [it]
- Pronunciation by lucaviolino (Male from Italy)
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Could you do it better? different accent? Pronounce coercizione in Italian
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- Random word: Atene
by Wiktionary Phonetic spelling:
by Yandex Translate English translation:
coercizione = coercion
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