text:
|Publication number
|Publication type
|Application number
|Publication date
|Mar 24, 1992
|Filing date
|Apr 19, 1991
|Priority date
|Apr 19, 1991
|Fee status
|Also published as
|Publication number
|Chia-Hui Shieh
|Original Assignee
|Beckman Instruments, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (5), Non-Patent Citations (4), Referenced by (48), Classifications (10), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention is generally directed to high performance capillary electrophoresis instruments and methods, and particularly to gel-containing microcapillary columns for high performance capillary electrophoresis.
Electrophoresis is one of the most widely used separation techniques in the biologically-related sciences. Molecular species such as peptides, proteins, and oligonucleotides are separated by causing these species to migrate in a buffer solution under the influence of an electric field. This buffer solution normally is used in conjunction with a low-to-moderate concentration of an appropriate gelling agent such as, for example, agarose or polyacrylamide, to minimize the occurrence of mixing of the species being separated. Two primary separating mechanisms exist: a) separations based on differences in the effective charge of the analytes; and b) separations based on molecular size.
The first of these mechanisms is generally limited to low or moderate molecular weight materials, such as, for example, oligonucleotides. This is because the effective charges of high molecular weight materials become rather similar, making it difficult or impossible to separate them.
Separations based on molecular size are generally referred to as molecular "sieving". Molecular sieving relies upon gel matrices having controlled pore sizes as the separating medium. In such separating systems, if the effective charge of the analytes are the same, the separation results from differences in the abilities of the different size molecular species to penetrate through the gel matrix. Smaller molecules move relatively more quickly than larger ones through a gel of a give pore size.
Oligonucleotides and medium-to-high molecular weight polypeptides and proteins are commonly separated by molecular sieving electrophoresis, although in the case of proteins, charge and size can be used independently to achieve separations. In the case of proteinaceous materials, however, it is generally necessary to modify the materials to be separated so that they all have the same effective charges. This is commonly accomplished by employing an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) derivitization procedure, such as discussed in Gel Electrophoresis of Proteins: a Practical Approach (2nd edition, B.D. Hames & D. Rickwood, Eds., IRL Press, Oxford University Press, 1990. See also, New Directions in Electrophoretic Methods, J.W. Jorgenson Ampersand M. Phillips, Eds., Published by American Chemical Society, Washington, D.C., 1987. Both of these references are incorporated herein by reference.
Occasionally, it is desirable to separate proteinaceous materials under conditions which pose a minimal risk of denaturation of the protein. In such cases, some additives such as urea and SDS, which can cause denaturation of the protein, are avoided. Because of this, the resulting separations are predicated upon differences in both the molecular size and charge of the constituents of the materials.
Most electrophoretic separations are presently conducted in slabs or open beds. These separations are very difficult to automate or quantitate. Extremely high resolution separations of materials having different effective charges has been achieved by open tube free zone electrophoresis and isotachophoresis in narrow capillary tubes. In addition, bulk flow (i.e., the flow through the capillary tube of the electrophoresis buffer and the sample being analyzed) can be driven by electroosmosis to yield very sharp electropherograms peaks. Such high efficiency, open tube electrophoresis has not generally been applied to the separation of medium-to-high molecular weight oligonucleotides, however, because these materials have very similar effective charges, as previously noted. Furthermore, open-tube electrophoresis does not provide size selectivity for proteinaceous materials. Achieving high resolution separations of, e.g., oligonucleotides, is directly related to the gel containing microcapillaries utilized in the electrophoresis system. Accordingly, and because of the importance of high performance capillary electrophoresis as a separating technique in the biological sciences, significant attention has been paid to the gel-materials which are utilized in this technique.
Polymeric gel materials typically employed in high performance capillary electrophoresis are usually any crosslinked polymer which has a pore structure which can be adjusted by varying the amounts of monomer and crosslinking agent, as well as the reaction conditions. Preferred polymeric gel materials are based on acrylamide and N'-methylenebisacrylamide, N'-methylenebisacrylamide serving as a cross linking agent. Other crosslinking agents include N,N'-(1,2-dihydroxyethylene)-bisacrylamide, N,N'-diallyltartardiamide, N,N'-cystamine-bisacrylamide, and N-acryloyltris(hydroxymethyl) aminomethane. Gel polymerization is typically initiated by ammonium persulfate and N,N,N', N'-tetramethylethylenediamine (TEMED).
The pore size of polyacrylamide gels is dependent on total monomer concentration (% T) and on the concentration of crosslinker (% C). Pore size can be progressively increased by reducing % T at a fixed % C; however, very dilute gels are mechanically unstable and pore sizes greater than 80nm cannot be obtained. The alternative approach is to progressively increase % C at fixed % T where the increase in pore size is considered to be due to the formation of a bead-like structure rather than a 3-D lattice. In this procedure, stable gels of high pore size (about 200-250nm) can be obtained at 30% C N'-methylenebisacrylamide; however, at higher concentrations, the gels become hydrophobic and prone to collapse.
While the capillary gels themselves have been rather well defined and analyzed in the art, preparation of the capillary tubes including such gels has not been well defined. For example, conventional gel-containing microcapillary columns maintain the capillary gel within the column by way of covalent attachment of the gel to the column. See, for example, S. Hjerte, "High-Performance Electrophoresis Elimination of Electroosmosis and Solute Absorption." J. Chrom., 347:191-198(1985); U.S. Pat. No. 4,865,707 (Karger et al) and U.S. Pat. No. 4,997,537 (Karger et al). The foregoing references are incorporated herein by reference. The gel-containing microcapillary columns described in the preceeding references suffer from at least the following problems: lot to lot reproducibility is unpredictable; the stability of the columns themselves is subject to failure after approximately 50-150 capillary runs; and the reaction time necessary for preparation of such columns can take as long as about 24 hours.
While capillary electrophoresis technology is generally directed towards the research environment, it has recently been suggested that this analytical technique has the potential for use as a clinical investigative technique. See "Capillary Electrophoresis: Tool for Clinical Diagnosis?", Clin. Lab Letter 12, 4: 1-2 (1991). While it is important in a research environment to ensure, e.g., lot to lot reproducibility and stability of the column, these concerns would be exacerbated in a clinical setting where patient analytical results require consistency from the analytical techniques utilized. Improved gel-filled capillary columns for electrophoresis which provides superior stability greater lot-to-lot reproducibility, improved resolution and shorter reaction times would be of great value to those fields that utilize such an analytical technique.
The foregoing needs are satisfied by the present invention which provides an improved gel-containing microcapillary for high performance electrophoresis. A gel-containing microcapillary according to the present invention includes a microcapillary, a bifunctional agent comprising at least one positively-charged amine and at least one active functional group, and a polymeric gel material. The bifunctional agent provides excellent adhesion of polymeric gel to the interior wall of the microcapillary. As used herein, the term "bifunctional" in reference to the agent means an agent capable of performing at least two functions, and is not a limitation as to the number of moieties included on the agent.
The bifunctional agent comprises at least one positively charged amino compound or a charged amino polymer and at least one active functional group. The positively charged amino is adsorbed onto the activated interior wall of the microcapillary. The active functional group is defined as a group including a carbon atom having at least one double bond. The active functional group is capable of copolymerizing with, e.g., acrylamide monomers. It, therefore, binds the gel material to the adsorbed bifunctional reagent.
Polyacrylamide gels which are coated to the interior capillary wall by the disclosed bifunctional agent require significantly less reaction time than gels which are covalently attached to the capillary wall. This is because adsorption, rather than covalent-binding, is utilized. Because the agent is adsorbed it, in effect, becomes a "part" of the interior wall of the capillary such that when the agent copolymerizes with the gel, the gel is securely affixed to the capillary.
Gel-containing microcapillary columns as disclosed herein may be prepared as follows: the interior surface of the capillary is first activated. The bifunctional agent is then introduced to the activated capillary whereby the positively-charged amine group is adsorbed onto the activated inner wall, and the activate functional group is exposed to the interior portion of the column. The microcapillary is next filled with a solution comprising at least one monomer with or without at least one crosslinking reagent; other materials, such as, e.g., a catalyst, may also be included in the solution. This solution is then allowed to polymerize within the microcapillary such that the polymerizing solution copolymerizes with the active functional group of the bifunctional agent. Ultimately, a polymeric matrix is formed which completely fills the microcapillary. By convention, at least one end of the gel-containing microcapillary is cut off cleanly and squarely, as a final step. The gel-containing microcapillary is then ready for use or for storage for subsequent use.
The following drawings are presented for purposes of elucidation in conjunction with the Detailed Description of Preferred Embodiments of the invention:
FIG. 1 provides a magnified perspective view of the end of a gel-containing microcapillary of the invention;
FIG. 2 is an electropherogram of the separation of a poly d(A) 25-30mer and a poly d(A) 40-60mer using 7M urea polyacrylamide gel-containing microcapillary in accordance with the present disclosure;
FIG. 3 is an electropherogram of the separation of the same oligomers of FIG. 2 using a polyacrylamide gel-containing microcapillary in accordance with the present disclosure;
FIG. 4 is an electropherogram of the separation of a poly d(T) 20-mer and a poly d(T) 40-mer using 7M urea polyacrylamide gel-containing microcapillary in accordance with the present disclosure; and
FIG. 5 is a graphic representation of the stability of a 7M urea polyacrylamide gel-containing microcapillary in accordance with the present disclosure as determined "Migration Time" vs. "Injections".
As depicted in FIG. 1, the gel containing microcapillary column in accordance with the present invention includes a microcapillary 10 including an activated layer 12; portion 12a is the inner most activated portion of microcapillary 10. Physically adsorbed to activated layer 12 is bifunctional agent 14 comprising a positively-charged amino compound or amino polymer 14a and an active functional group 14b; active functional group 14b is copolymerized with a polymeric gel 20 within the bore of microcapillary 10.
The bifunctional agent 14 has the following chemical structure: ##STR1## where three of R1, R2, R3 and R4 can be independently selected from the group consisting of ##STR2## where n is an integer between 0 and 10;
m is an integer between 0 and 5;
R is selected from the group consisting of ##STR3## the proviso being that at least one of R1, R2, R3 and R4 must comprise an active functional comprising a moeity selected from the group consisting of ##STR4## y is an integer between 1 and 5000, more preferably between 1 and 2000, and most preferably 1; and
X is selected from the group consisting of flourine, chlorine, bromine, iodine and astatine.
In a most preferred embodiment of the bifunctional agent R1, R2 and R3 are each -(CH2)n CH3 where n is equal to 0 (i.e. -CH3), R4 is (CH2)2 =CH2, X is iodine ("I") and y is 1. Accordingly, a most preferred embodiment of the bifunctional agent can be represented as follows: ##STR5##
The particular materials used in the manufacture of microcapillary 10 are well known to those in the art and are typically selected with the proviso that the particular detection systems utilized in the electrophoresis can adequately function with such material. Such materials include, e.g., glass, alumina, beryllia, fused silica, and TEFLON. Preferably, the microcapillary is made of fused silica. Preferably, the internal diameter of the microcapillary is between about 10 and about 2000 microns, more preferably between about 10 and about 200 microns, and most preferably about 100 microns.
The interior portion 12a of microcapillary 10 is pretreated prior to introduction of the bifunctional agent thereto. Such pretreatment "activates" interior portion 12a, thus providing a negative charge thereto. Pretreatment is preferably accomplished by filling microcapillary 10 with lN HCl for 10 minutes, followed by lN NaOH for 10minutes, followed by deionized waterwashing for 10 minutes. This standard procedure efficiently activates the microcapillary for adsorption purposes.
The agent is introduced to the capillary as a bifunctional agent solution. The bifunctional agent solution is most preferably an aqueous solution. However, polar solvents such as, for example, dioxane, dimethylformamide, acetane, methanol, or ethanol may be used. Preferably, the solution contains between about 1% and about 80% (by volume) of the bifunctional agent, more preferably between about 10% and about 50%, and most preferably about 30%. The pH of the bifunctional agent solution is preferably between about 4 and about 12, more preferably between about 5 and about 9, and most preferably about 8. However, when any of R1, R2, R3 or R4 is/are H, then the pH of the bifunctional agent solution is less than about 7.0 so that protonation of the amine can occur.
The bifunctional agent solution is then introduced into the activated microcapillary and flushed through it for between about 1 minute and about 60 minutes, more preferably for between about 15 minutes and 45 minutes, and most preferably about 30 minutes. After this period of time, the bifunctional agent is adsorbed to the microcapillary. Preferably, adsorption takes place at about room temperature, although temperatures of 20-35� C. are acceptable. After the bifunctional agent has reacted with the microcapillary inner wall, the inner portion of the microcapillary is ready for introduction of a polymeric gel material thereto.
Polymeric gel materials applicable to capillary electrophoresis are well known. The polymeric gel material 20 utilized can be any polymer which has a pore structure which can be varied--the gel may or may not be crosslinked. Preferably, the polymeric gel is a crosslinked polymer whose pore structure is varied based upon the amounts of monomer and crosslinking agent added to form the gel, as well as varying the reaction conditions. As previously indicated, the variations in the total concentration of the monomer and the crosslinking agent are generally expressed as % T and % C, respectively. For the monomer acrylamide and the crosslinking agent N,N'-methylenebisacrylamide system which is most preferably utilized in the invention, % T and % C are derived as follows: ##EQU1## See, Hjerten, Chromatographic Review 5, 9:122-219 (1967) which is incorporated herein by reference. The concentrations of monomer and any crosslinking agent are determined according to the porosity of the polymeric matrix desired. As noted previously, the reaction is preferably initiated with ammonium persulfate. However, the concentration of initiator (and polymerization catalyst, if any) in the reaction mixture can be determined experimentally. This can be accomplished by preparing test solutions containing the desired % T and % C, but varying the amount of initiator (and polymerization catalyst) utilized. Preferably, % T is from about 1 to about 30, more preferably from about 3 to about 15, and most preferably about 12. Preferably, % C is from about 0 to about 15, more preferably from about 1 to about 6, and most preferably about 2.
After % T and % C have been determined, a fresh mixture of polymerization mixture is prepared and injected into the microcapillary tube, typically by means of a syringe. The formation of bubbles is to be avoided as this can lead to a useless gel-filled microcapillary column. When the microcapillary tube has been filled with the polymerization mixture, the ends of the microcapillary are plugged or capped, and such plugging or capping is maintained on the microcapillary while the polymerization reaction occurs.
The polymerization reaction is most preferably carried out at about room temperature and for at least about 2 hours, more preferably overnight. However, polymerization temperatures and times can vary depending on the gel material utilized.
As a final step, after the polymerization reaction is completed, the plugs or caps are removed from the ends of the microcapillary and at least one end of the microcapillary is cut off cleanly and squarely. The object of such cutting is to leave a smooth surface of gel material exposed at the end of the microcapillary--it is to this end that the sample to be analyzed can be introduced. The gel-filled microcapillary column may then be placed in suitable electrophoresis apparatus. The column can be checked on the apparatus by applying a very low electric field (about 100 to 150 volts/cm) for about one hour to the capillary. An improperly prepared column will generate a "noisy" baseline or a zero current condition.
High resolution capillary electrophoresis using the prepared gel-filled microcapillary columns includes the steps of electrophoretically injecting an aliquot of a sample containing constituents to be separated into the column of the invention, applying an electric field of between about 50 and about 600 volts/cm, allowing a current preferably less than about 2 to about 50 microampers to pass through the microcapillary, and instrumentally detecting and measuring the separated analytes sequentially as they migrate past an on-line detector. Typically, the measurements are presented as electropherogram peaks, where each sample constituent that is detected has a defined peak based upon the flow rate of the constituent past the detector as well as the amount of the constituent in the sample.
The improved gel-filled microcapillary columns containing a bifunctional agent comprising a positively charged amine and an active functional group exhibit excellent stability and have resolving power (i.e. the ability to separate the sample constituents) equal to or better than previous columns. Additionally, such columns exhibit excellent run-to-run, batch-to-batch reproducibility. The present columns require only about 30 minutes for preparation of the adsorbed-bifunctional agent microcapillary, as opposed to approximately 24 hours required for covalently attaching materials which bind the gel to the microcapillary.
The following examples are presented for illustrative purposes only and are not intended, nor should they be construed to be, a limitation on the foregoing disclosure or the claims to follow.
Capillary electrophoresis of the samples described in the following Examples was performed on a Beckman Instruments, Inc. PACE™ high performance capillary electrophoresis system (Beckman Instruments, Inc., Fullerton, CA. Model No. #2000). Data analysis was performed on System Gold™ software (Beckman Instruments, Inc.). The PACE™ high performance capillary electrophoresis system contains built-in 214, 254, 280 and 415nm narrow-band filters for on-line detection. The detection window was located approximately 6.5 cm from the column outlet.
Samples were placed on the inlet tray of the above-referenced capillary electrophoresis system. Samples were automatically injected into the gel-filled microcapillary tube by the electrokinetic method for 1.5 seconds at 75 kV.
The electrophoresis buffer was 7M urea in 10mM tris-hydroxymethyl amino methane--250mM borate, pH 8.3.
106 g of sodium carbonate (J. T. Baker, Phillipsburg, N.J., Cat. #3604), 37.5ml of alkyl amine (Aldrich Chemical, Milwaukee, Wis., Cat. #24107-5), and 200ml of methanol (B&J, McGaw Park, Il., Cat. #230) were stirred in a lL round bottom flask. 124.5 ml of iodomethane (Aldrich, Cat. #I-778-0) in 50ml of ethanol was slowly added to the flask by a dropping funnel. The reaction mixture was stirred overnight (18 hours). The methanol was evaporated on a rotavap. 15 ml of iodomethane was then added to the flask, followed by 10 minutes of shaking. 200 ml of water and 20 ml of tetrahydrofuran (B&J, Cat. #340) was added to the solution and stirred for 20 minutes. The sodium carbonate was then filtered, and the aqueous layer was collected and filtered through a 0.2 micron membrane filter.
The resulting solution included a quarternary alkyl amine bifunctional agent.
A 65 cm, 100 micron i.d. microcapillary column (Polymicro, Phoenix, Ariz., #TSP100375) was pretreated by introducing to the interior of the microcapillary 1N hydrochloric acid for 10 minutes, followed by 1N sodium hydroxide for 10 minutes, followed by a deionized water rinse for 10 minutes. After pretreatement, the gel column was coated with the bifunctional agent of Example I by placing the solution in the capillary for 30 minutes.
For 7M urea gel, 1.2 g of ultrapure acrylamide (ICN, Irvine, Calif. Cat. #814320) was dissolved in 10 ml of 7M urea, tris-hydroxymethyl amino-methane-borate buffer ("tris-borate" buffer) (7M urea, 100 mM tris, 250 mM borate, pH 8.3). The acrylamide solution was then filtered through a 0.2 micron nylon filter. 2ml acrylamide solution, 2 microliters of 10% ammonium persulfate (BRL, Gaithersburg, Md. Cat. #5523) in deionized water, and 2 microliters N,N,N',N'tetramethylenediamine (BRL, Cat. #5524 UB) were mixed and poured into the coated microcapillary column using 10 psi helium gas; after 31/2 minutes the pressure was decreased to 5 psi followed by continued, gradual decrease such that after 5 minutes the pressure was 0 psi. The microcapillary ends were capped and stored at room temperature for 18 hours for polymerization. The gel-filled microcapillary tubes were then stored until used at 4-8� C.
For a gel column not containing 7M urea, the foregoing procedure was followed, the exception being that the acrylamide was dissolved in tris-borate buffer (100mM tris, 100mM borate, pH 8.3).
Poly d(A) 25-30mers and 40-60mers (Pharmacia, part #27-7986-01 and #27-7988-01, respectively) were separated on the aforementioned PACE™ high performance capillary electrophoresis system using the 7M urea gel-filled microcapillary column of Example II. The microcapillary had a 47 cm total length, 40 cm effective length and an i.d. of 100 μm. The injection was accomplished with 75 kV for 1.5 seconds of 1.50 OD units/ml of sample. The analytical run was accomplished with 11.1 kV at 30� C.
Analytical results are presented in FIG. 2 where absorbance (�10-3) is plotted against the horizontal axis, migration time is plotted against the vertical axis and the poly d(A) 25-30 mer appears in time before the poly d(A) 40-60 mer.
The same oligomers of Example III were separated in accordance with the procedure of Example III using the non-urea gel-filled microcapillary column of Example II. Analytical results are presented in FIG. 3 in accordance with the description in Example III.
A mixture of poly d(T) 20 mer and 40 mer (Pharmacia, part #28-8610-01) was separated in accordance with the procedure of Example III using the 7M urea gel-filled microcapillary column of Example II. Analytical results are presented in FIG. 4 in accordance with the description of Example III, the description being that the poly d(T) 20 mer appears in time before the poly d(T) 40 mer.
Migration times for poly d(A) 25, 29, 41, 47 and 50mers were obtained across a number of injections of the oligomers using the 7M urea gel-filled microcapillary column of Example II. Results are depicted in FIG. 5. The results demonstrate that the gel-filled microcapillary column is stable for at least about 100 injections. In FIG. 4, the following symbols are used: poly d(A) 25-� ; poly d(A) 29-• ; poly d(A) 41 x; poly d(A) 47 - x; and poly d(A) 50 - /.
The bifunctional agent of the present invention provides for adsorption of the amine-portion of the reagent to the microcapillary within about 30 minutes after the agent is introduced thereto. The active functional group is then capable of copolymerizing with the polymerizing gel material added to the microcapillary. The foregoing examples demonstrate that a gel-filled microcapillary column incorporating the bifunctional agent has excellent resolving power and are stable for at least about 100 injections. The bifunctional agent requires only about 30 minutes for adsorption to the capillary inner wall, as opposed to about 24 hours required by the prior art covalent attachment. Accordingly, the present bifunctional agent offers several additional advantages over previous gel-filled microcapillary columns.
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|U.S. Classification
|204/453, 204/455, 204/604, 204/605
|International Classification
|Cooperative Classification
|European Classification
|Apr 19, 1991
Owner name: BECKMAN INSTRUMENTS, INC., A CORP. OF DE.
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Saturday, 19 June 2010
How To Be A Good Friend
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subset: commoncrawl


text:
Accessibility on public transport
What more can be done to improve accessibility on London's public transport (bus/underground/overground/DLR/river transport etc) for elderly and disabled people and parents with pushchairs?
Transport is part of daily life for anyone who lives or works in London. 30 million journeys were made on the Transport for London network each day in 2015, and we walk, cycle, drive and take minicabs around our city too.
A current priority issue for the capital is the need for cleaner air. We need to reduce emissions from public transport and make a transition to less polluting cars and minicabs. Change is needed on a local level, both in central zones and through the rest of London, particularly along routes children walk to school. There's currently worse air quality in some of the most deprived areas of our capital – we need to act to remove these inequalities.
How do we provide a fairer, more accessible and more environmentally-friendly travel network?
Join in with a discussion below or start up your own.
To start a discussion please register or log in
What more can be done to improve accessibility on London's public transport (bus/underground/overground/DLR/river transport etc) for elderly and disabled people and parents with pushchairs?
I believe that those who can afford travel shouldn't have a freedom pass, paid for by workers who earn less, or the unemployed. The over 60's with money then take the seat of the exhausted worker.
Could we please have eco friendly, electric, hybrid or fuel cell hire vehicles in London as I'm fed up with the lack of choice here and the stinky foul toxic air we have to breathe in from the usual polluters nobody in power is doing anything REAL about in 2107!!!!
This is a radical idea, but might just reduce traffic more than any congestion charges or other plans. Why not add a transport surcharge onto all council tax bills and give London council tax payers a free Oystercard like the Freedom pass?
As I was crossing the North Circular's A406 today at around 6.20pm whilst the ongoing traffic stopped to obey the red signal lights, to my shock, I suddenly noticed before me a motorbike zooming pass on the bus lane, jumping the red lights on this popular pedestrian crossing.
Should London implement jaywalking legislation in a similar at to the United States, Austria etc. Pedestrians stepping out wherever and whenever they wish is a risk to both themselves and other road users and it slows traffic flow.
Hello All.
An idea was floating around my household a few weeks ago of installing hand sanitiser dispensers at London Tube Stations to prevent virus/colds etc.. They now have them at most gyms which seems to be working well.
Should public transport costs be directly linked to air quality so that when pollution is at its worst public transport costs should be reduced to encourage more people to use public transport rather than the current policy of peak and off peak fares?
I have across more than 50 incidents where cars have been going through red lights and the green man is flashing. It is so dangerous for pedestrians while crossing.
To get a bus from lower down TCR to Regents Park or Madame Tussauds or Baker St you have to get 2 buses.
Dispite the serious concerns of pollution poisoning London's atmosphere and subsequently ruining the public's health, there does not seem to be a decrease of private car use during the day on our roads, in fact traffic brought about by private cars usage seem to be increasing.
Apart from clean Air and Clean Streets we also need Clean Buses.
I have observed for many months that the exterior of our London Public Buses are filthy, completely covered in dust.
The line is probably the worst in london in the morning. There are signal failures every week or two for 2 to 3 days. These so called minor delays mean when you wait for a tube you cannot get on as it is full of heathrow passengers arriving.
Takeaway carton, chicken bones, half empty drink cartons over the floor of the bus and at times on seats. the bus driver says that their option is to terminate the bus at the time of the incident. individuals swiping there hands on the fabric of seat after eating takeaways.
To stop large parts of the boroughs of Barnet and Camden being forced in to gridlock, I feel it is about time TFL throw out plans for a cycle superhighway at Swiss Cottage.
The widening of London's pavements, reduced traffic lanes and the disappearance of the recessed bus-stop bays years ago, have definitely contributed to the worsening of the traffic.
Why is that cyclists in London frequently abuse traffic rules and regulations and the Government turn a blind eye?
Why is it that cyclists don't
pay road tax?
have registration?
or are required to pass a cycling proficiency examination?
I would like Wandsworth to be linked with Fulham by a direct bus service that doesn't go via Clapham Junction and involve changes.
Streatham/Tooting Broadway, Tooting Bec, Trinity Road, Wandsworth Bridge, Fulham. (on to Shepherd's Bush?)


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50 to 1. It's a cute little place where folks can submit a fifty word story or the first line of a story. Today I was published there. This will be my second time. :) Go take a look.
(This just in. My niece has discovered that a word is missing from my one liner. Oh the humanity!)


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UNC-Greensboro Snares Parkland Guard
At the end of the high school basketball season in North Carolina, Ray Bristow was playing at the top of his game. He was routinely scoring points and Parkland was winning.
The team with double-digit losses (many of which came early in the year) was on a roll and Bristow was spearheading the charge. The point guard averaged 18 points and 5 assists this season.
Until Tuesday, his recruitment had been kind of slow. Now, he's committed to the UNC-Greensboro program. Stetson and some others took a look, but the Spartans got it done.
Greensboro and Bristow is an excellent match for each other. Bristow's Parkland teammate, shooting guard Mark Mason, will walk on for the Spartans.


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For the past few months I've been working on a definitional essay of the Emerging Church Movement (ECM), mostly because as an Americanist in religious studies and anthropology, I would like to know, in terms of the production of classificatory academic knowledge, (i) what the ECM is, (ii) where the movement originates, (iii) what it is like, descriptively, and (iv) where it fits in the spectrum of emerging North American Protestantism (notice: lowercase c).
That the ECM resists definitions, that it defies etic categorization, is by now truistic in every sense of the word. The ECM is diverse—there's no question at all—and its practices and rituals vary per community or gathering. I concede the point. But enough primary source monographs have been written by emerging writers to begin the comparative program that is the core of religious studies. Emic definitions are ripe for comparison and contrast. Myriad blog posts bombard online discourse hubs, just waiting for analysis. As of yet, scholars have all but ignored the academic study of the ECM, with the masterful and recent exception of anthropologist James Bielo's ethnographically multi-site Emerging Evangelicals.
At this point, I'm still collecting definitions and formulating my own. I've consulted the writings of many of the movement's primary writers: Tickle, Jones, Ward, Knight, McLaren, Pagitt, Gibbs and Bolger, McKnight, Snider and Bowen, Rollins, and Scandrette, just to name a few. But as I've reflected on the diversity of the movement, especially as represented by these above figures, I've had a thought: Emergents—or those emerging, or those who self-identify with the ECM—are dirty evangelicals. Here I draw on anthropologist Mary Douglas' famous theory of taboo and pollution, dirt and order. As she puts it in her own words, dirt is "a kind of compendium category for all events which blur, smudge, contradict, or otherwise confuse accepted classifications." Dirt blends and smears categories; it blurs and obfuscates delineations. And while the term is certainly vulgar and possibly offensive at first glance, it does seem to instructively describe the state of the ECM. Emergents defy both academically descriptive as well as theologically-derived taxonomies. I'm thinking here of the ECM's close identification with all things "postmodern," which in some fields of the academy, most notably the visual arts, might be described in terms of hyper-eclecticism, combinativity, and juxtaposition. I'm thinking here of Tickle's claim in The Great Emergence that emerging communities blend together the four quadrilaterals of Christian practice (which she calls the "Liturgicals," "Social Justice Christians," "Renewalists," and "Conservatives"). I'm thinking of McLaren's multifarious A Generous Orthodoxy.
One more point reinforces my use of Douglas' theory to categorize the ECM. Within conservative evangelicalism, the reaction to this point has been primarily negative. Many see the ECM as theologically dangerous, and so construe it, unknowingly, of course, in Douglas' theoretical terminologies. The ECM, to much of evangelicalism, operates at the margins of traditional Christianity (i.e., it is marginal) and its teachings heterodox. It operates at the fringes of Protestantism. Its practices are polluting.
So, what do you think? Are emergents "dirty" in the way Mary Douglas envisioned the term?


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Coco's Sunset Grille Spring Special
Monday: Snow Crab Night!
Two clusters (over a pound) with coleslaw and corn for $18.95. Add a cluster for $6.95.
Tuesday: Trivia Night!
Starts at 7pm. Winning team receives a $25 CoCoCard.
Wednesday: Sushi, Sake & Sunset!
Try one of our original sushi rolls along with different varieties of sake or our Sake Sunset Splash!
Thursday: Tapas Thursday!
International Food and Drink Specials!
Friday: Low Country Boil Night!
Local shrimp, corn, sausage, potatoes all cooked together and served family style.
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Fun and games throughout the day for bar prizes!
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Along with a yummy new Hangover Menu!
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For more information about Coco's Sunset Bar & Grill click here.


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CANP is a major development focus of the international bodies of mathematicians and mathematics educators (International Mathematical Union, IMU & International Commission on Mathematical Instruction, ICMI) in conjunction with UNESCO and International Congress of Industrial and Applied Mathematics, ICIAM. The project is a response to Current Challenges in Basic Mathematics Education (UNESCO, 2011).
CANP aims to enhance mathematics education at all levels in developing countries so that their people are capable of meeting the challenges these countries face. It will develop the educational capacity of those responsible for mathematics teachers, and create sustained and effective regional networks of teachers, mathematics educators and mathematicians, also linking them to international support.
CANP consists of an ongoing series of Programmes in a different developing region each year.The first programme CANP 1 Edi Math was held in Mali with participants from across Sub-Saharan Africa in September 2011 and was supported by UNESCO, CIMPA and ICIAM. CANP 2, Central America and the Caribbean was held in Costa Rica in August 2012. It was supported by the Regional Office for Latin America and the Caribbean (ROLAC), UNESCO, ICIAM and CDC. The third CANP 3 South East Asia was held in Cambodia in 2013 and was supported by CDC, RECSAM, SEAMS and CANP 4 East Africa was held in Tanzania in September, 2014. CANP 5 Andean Region and Paraguay will be held in Lima, Peru in February, 2016.
Each programme has, at its centre, a two-week workshop of about forty participants, half from the host country and half from regional neighbours'. It is be primarily aimed at mathematics teacher educators, but each programm will also include mathematicians, researchers, policy-makers, and key teachers. Each workshop has associated activities such as public lectures, satellite workshops for students, or exhibitions reaching out to a board audience.
The coordination of the workshop is undertaken by a group of eight (IPC) with equal participation by mathematicians and mathematics educators from the region and from the international IMU/ICMI/ICIAM community. The Local Programme Chair and the Programme Coordinator are members of the IPC. Each member of the IPC has to come from another country. The IPC and the Local Programme Chair are appointed by the ICMI EC.
The Programme Coordinator is appointed by the ICMI EC in consultation with IMU.
After the appointment of the Local Programme Chair, the Programme Coordinator and the IPC, the Local Organising Committee (LOC) is appointed by the Local Programme Chair, the Programme Coordinator/ Manager (in agreement and consultation with the ICMI-EC).
The Local Organising Committee consists of the national/ country contact person (one each per participating country) and members of the host country. The LOC selects in together with the Programme Coordinator/ Manager the participants for each CANP programme.
The program, and in particular the workshop, builds on existing activities in the region and does not seek to reproduce or compete with existing development programmes. It includes a follow-up after one year to ensure continuity and for evaluation. A post-workshop and a 1-year report are produced for each programme. ICMI will ensure that the experience of earlier programmes will be used for the design of later ones.
The Capacity & Networking Project is a major international initiative in the mathematical sciences in the developing world to help exchange information, share the state of the art research, and identify strategies for addressing barriers to enhance mathematics education and to conduct to building a sustainable network for policy makers, scholars and practitioners across those targeted regions.
CANP Review
The Issue
There have been four CANP Workshops spanning 6 years.
ICMI has been spending a lot of money funding the CANP project. The initial conception had an evaluative component built into each CANP that has really not happened in any formal way. Is it cost effective? What else, or what differently, could we do?
Our experience so far has shown us that the different regions have responded differently to the CANP opportunity, and that individuals make a huge difference one way or the other.
ICMI EC is receiving legitimate calls for funding for follow-up activities from each CANP and needs to decide on a sensible, adequate and sustainable response. With limited resources and other eligible regions, we need to decide how best to spend the available money to both ensure the money already spent has maximum effect, but also to meet global responsibilities.
How best can the aims of CANP be met?
CANP Review Proposal
A proposal to review CANP has been presented to the ICMI EC and been accepted at the EC meeting in Macao in June 2015.
It was proposed to review of CANP as a whole (rather than individual evaluations of each CANP) for which a Review Committee should be established. The committee members are Michèle Artigue and Bill Barton (as the originators and CANP Managers of the programmes in Mali and Cambodia), Angel Ruiz (as one of the CANP ICMI Liaisons), Ferdinando Arzarello (as ICMI President as well as being a CANP ICMI Liaison), and Lena Koch (for her knowledge of all five CANPs, her international development experience, and her financial management experience). Additionally Abraham Arcavi as ICMI SG is invited as ex-officio of the Review Committee.
The following Terms of Reference were proposed:
· Evaluate the whole CANP programme for how well it meets the original aims and its other impacts.
· Evaluate the whole CANP programme for value for money and financial efficiency and sustainability.
· Review and update the aims, description and criteria documents for CANP.
· Make recommendations on how existing CANPs are further supported.
· Make recommendations on new CANPs, or other CANP activities.
Lena Koch is coordinating the process of the CANP review.
As a first step during January and June 2016 the participants, organizers and IPC member answered two online surveys regarding their CANP experiences.
The first results of the surveys were presented at the EC meeting in Berlin in July 2016. The presentation can be found here.
In July 2016 an ICME13 discussion group had two fruitful meetings regarding CANP and its future.


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Tagsadaptil Agression Blog broadline cat cats cestem Conflict dog dogs dog wormer drontal feliway Feliway Friends fireworks fleas frontline Frontline Petcare granofen home horse horses John The Vet Joints Kittens moving panacur Pet Blog pet care Pet Food Pets pet tips Safety solution spot on stress Summer ticks vet advice vet medic wormers worming worming tablets worms zylkene
Lily's Kitchen Pet Food Update (April 2017)
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Keeping Your Pets Winter Safe With Frontline Petcare
We are a nation of animal lovers, with up to 99% of owners considering their pet to be a member of their family – 9% even have their own Facebook page! Watching our pets' racing around care-free on a walk or chasing a toy, brings a smile to our faces. We get so much pleasure from our furry companions and … Continue reading
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The Benefits Of Doggy Dental Treats & Toys
Caring for our teeth is easy but for dogs, who cannot brush their own teeth, support from us is required. Dogs do not have a natural way to keep their teeth clean and thus, it is up to us to ensure that they are clean and healthy. When it comes to our dog's health we sometimes forget about the importance of … Continue reading
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12 Christmas Pet Dangers
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Top 5 Halloween Pet Safety Tips
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Chac: The Rain God is Rolando Klein's obviously-peyote-influenced tale of Mexican Indians desperate for rain to water their crops. The tribal farmers chant and consult the local shaman, but to no effect. Desperate, they pay a visit to the diviner, who lives up in the mountains, and he takes them on an epic journey culminating in a headdress-festooned rain dance.
Continue reading: Chac: The Rain God Review
The Stooges frontman Iggy Pop awarded France's 'highest honor'.
Guns N' Roses have grossed $230m from their 'Not In This Lifetime' tour so far.
Jason Drucker is Greg Heffley in the upcoming movie based on the novel of the same name.
Ford attributes his career success to films that pass 'from generation to generation'.


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Latest Stories
Black Tiger Review0 Comments
February 4, 2011
Classic Capcom Arcade Titles Coming to Wii's Virtual Console Arcade0 Comments
November 8, 2010
Help us solve the toughest (or most puzzling) game puzzles and challenges.
Our attempt to answer issues which most commonly occur on IGN's wikis.
About This Game
- Summary
- Specifications
- Game Editions
This downloadable game is based on the original arcade version of this classic game, provided through Nintendo's Virtual Console service.
A barbarian hero jumps and fights his way through a variety of colorful, enemy-packed levels in this spiritual successor to the Capcom classic "Ghost 'n' Goblins". The sprawling, eight-way scrolling levels are packed with hidden bonuses to encourage and reward exploration. Most of these bonuses are in the form of 'Zeny coins'; currency that allows the player to buy such items as an upgrade to their weapons and armour, keys for treasure chests, and anti-poisoning potions. Special items that reveal coins, upgraded armor, or simply bonus points may be found by attacking certain walls.
- Supported Functions
- Number Of Players: 1
Black TigerPublisher: CapcomRelease Date: January 24, 2011MSRP: 8 USD
Black DragonMSRP: 800 JPY


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Question… What's the Biggest SEO Challenge?
Answer is……
Building quality Backlinks.
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Increasing Your Email Reply Rate
Moz recently came out with a great article titled
How We Increased Our Email Response Rate from ~8% to 34%
It's no secret that reply rate is the golden metric of email campaigns.
The reason is obvious. As opposed to open and click rate, reply rate tracks how many recipients were interested (or annoyed) enough to actually write you back. For guest blogging and email outreach, your reply rate will determine your campaign's success.
We still believe that guest blogging is a great opportunity to improve your site's link profile and brand exposure. However, the time-investment needed in prospecting/email outreach can leave you questioning its ROI….
Read the entire blog here….
Welcome to the new Design ME website!
They say "The shoemakers shoes are never fixed."
Not sure who "they" are, but there is truth. We are working on so many projects that our own site is slowly being launched. Check back here for great content….. hopefully soon.


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// Emma Cowley
'If I was Ira I would join the circus with my troupe of performing cats!'
// www.emmacowley.co.uk // Emma's blog
◆ Question time
Owls or Eagles?
Eagles! Nah just kidding, owls it is... but you probably already guessed that, judging by how much they show up in my illustrations!
Who or what is your biggest inspiration?
I respect a lot of different artists, but the natural world, plants, and animals are the root of all my work.
Would you really trade your life with someone else?
Could I choose who to trade it with, like someone who's filthy rich? Ha ha, no I'm happy with what I have... and after all, money cannot buy you happiness!


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28 February 2011
MT Musical instruction and study
781 General principles & musical forms
Borland, D. The Singer's Psyche: a psychological approach to vocal performance training. (Thesis). Edinburgh Napier University. Retrieved from http://researchrepository.napier.ac.uk/id/eprint/4529
Voice performance training; well-being; mental health; physical fitness; psychological impacts; power differentiation; communication;
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In Reply We concur with Dr Hehir and colleagues that a wide range of clinicians can provide high-quality, patient-centered, prenatal genetic counseling and that women considering invasive diagnostic testing usually meet with a trained professional to discuss risks and benefits prior to providing informed consent. However, because most pregnant women do not undergo invasive testing, they do not meet with such specialists.
More commonly, women receive brief explanations of aneuploidy screening during their first or second prenatal visit, with little time to engage in nuanced shared decision making. We believe that such women could benefit from better information on their options and an opportunity to clarify their values, and that a decision-support guide can help ensure more informed decision making. Our intention was not to suggest that a computerized decision-support guide should replace genetic counseling. Rather, our goal was to gain an understanding of the decisions women from a range of sociodemographic backgrounds would make if they were fully informed about screening and testing options, their voluntary nature, and their potential outcomes, and did not face financial barriers to testing.
Kuppermann M, Norton ME. Decision-Support Guide and Use of Prenatal Genetic Testing—Reply. JAMA. 2015;313(2):200-201. doi:10.1001/jama.2014.16488
© 2017


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Your Index to News about
Northwestern University Athletics
Men:WOMEN's BASKETBALL
Wildcats Fall To No. 21 Badgers, 68-52
+ Wildcats get Badgered
+ Cursed at Kohl: NU 52 Wisc 68
+ Clanking Cats fall on road
+ Badgers adjust, handle Wildcats
+ Late 13-2 first-half run dooms NU
+ Badgered into submission
+ Badgers humble Cats
Coach Roy: Random Thoughts: Bad O or Good D?
Shea: Can Cats make NIT?
From Wisconsin
Badgers Trounce Wildcats, 68-52
+ Notebook: Frosh contain Vukusic
+ Tucker's 20 points lift UW over Northwestern
+ Rob Schultz: Ryan plays it smart with simple, consistent approach
+ Patient Badgers skin Cats
Next Up Michigan
Other Big 10
MSU's Davis Nabs Men's Basketball Weekly Laurels
Senior duo saves day for Illinois
+ Illini defense shows chinks in its armor
Purdue bounces back for Painter's first Big Ten win
Short break for Indiana OK by Weber
Haluska finds touch at good time for Iowa
+ Haluska, Iowa win at Penn St.
'Cats Bested by Hawkeyes, 80-48FOOTBALL
+ Second-half surge turns early lead into blowout
+ Hawkeyes exploit Cats' shooting woes
+ Hawkeyes finish satisfying stretch with blowout win
Post Season All Star Games:RECRUITING
East West Shrine: Basanez, McGarigle, Cofield
Newman Speaks OutSWIMMING
Chicagoland hoss high on Beavs
Oliver Confirms OSU Scholarship Offer
Browning Gives The Latest
Do assistants bring players?
Cooks delivers two commitments from Texas
No. 17 Wildcats Finish Second at Dallas Morning News Swimming & Diving Classic
Wildcats Take Five-Straight Singles Matches to Top Ball State, 5-2
Cristelle Grier and Alexis Prousis Finish Second At National Collegiate Tennis Championships
+ Wildcats Earn Two Berths In Doubles Semifinal Round at National Collegiate Tennis Championships
13th-Ranked Wildcats Fall to No. 18 Arizona State, 22-17MISCELLANY
+ Herbert still undefeated, but NU knocked out early
Clarett victim of entitlementAlumni
Bentley [Seahawks] and Essex [Steelers] advance
The Original Northwestern URL Link Service
Coach Roy's Comments on the stories are in Italics:
Remember that I try to only get one copy of an AP story.
$ before a story means its from a source requiring a subscription.
Opinion Columns usually have the name of the writer in bold at the front of the headline.
[Click Here]
"All the News that Fits we Link"
Copyright 2006 Purple Reign/PurpleWildcats.com
div of Computer Applications & Support Associates, Inc.


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Need a book? Engineering books recommendations...
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Re: Blast Door Design : Edge Rotation[Subject Prev][Subject Next][Thread Prev][Thread Next]
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- Subject: Re: Blast Door Design : Edge Rotation
- From: Himanshu Goyal <anshuagoel(--nospam--at)gmail.com>
- Date: Mon, 29 Oct 2012 13:36:05 -0500
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Hello: Which provision in UFC manual sets this limit. Could you please post a link for it. On Mon, Oct 29, 2012 at 1:10 PM, Kapil Nandwana <kapil.nandwana(--nospam--at)gmail.com>wrote: > Hi, > > I am currently working on a blast door design and geometry of the door > and pressure requirements are such that despite having a ductility ratio > of less than 1, i.e. door remains in elastic region, I am getting a edge > support rotation angle greater than 2 degrees (which is the limit set in > UFC manual for protection category-I structure). > > My question is, since I remain in the elastic region, there is no way the > blast door is going to fail. In that case, should I be worried about not > meeting the support edge rotation criterion. This edge rotation criterion > seems to be an overly conservative criterion. > > Thanks, > > Kapil ------------------------------------------------------------------------- Post your message to the list by sending it to: SEAINT-SEAOSC(--nospam--at)mail-list.com. The email messages sent to the list will be saved in an archive on the World Wide Web. These archives are located at: http://archive.mail-list.com/SEAINT-SEAOSC To contact the list owner, send your message to: SEAINT-SEAOSC-list-owner(--nospam--at)mail-list.com. 5700 Ralston Street, Suite 300, Ventura, CA 93003 To unsubscribe, switch to/from digest, get on/off vacation, or change your email address, click here. http://cgi.mail-list.com/u?ln=seaint-seaosc&nm=seaintma%40euken.net
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It's Really F*cking Hard To Understand Sofia Vergara In These Bloopers (Video)
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It's not so easy to understand what Sofia Vergara is saying.
She has a thick Colombian accent and is known to speak at lightning speeds.
But she's at least able to make fun of her linguistic shortcomings.
In this blooper reel for her new film "Hot Pursuit," you can hear just how difficult it can be to decipher Vergara's words.
I think Vergara has given Iggy Azalea a run for her money when it comes to spouting out pure gibberish. See what I mean above.
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CITATIONS Bustle


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Mid-century and unusual smaller sized vintage haberdashery counter in light beech wood. Glazed to 3 sides and the top. 12 graduated drawers.created by: Withy Grove Ltdorigin: Leeds, UKyear: 1960dimensions: width: 120cm; height: 99cm; depth: 58cm
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I have a midterm tomorrow. I should be studying. But am I? No. I'm writing PruCan fluff. And when I say fluff I mean full-on fluff. This is pretty much what happened in a dream I had the other night, except things took a turn for the worse and people died. I thought the beginning was still decent though, so what did I do? I made them live happily ever after and stuffed in as much fluff as I possibly could. It's what I do. It's what I live for. Join me in the squee-fest! I hope you all enjoy it!
Matthew curled up on the couch as thunder crashed outside. He could hear his boyfriend Gilbert cackling madly as he ran through their house with his little yellow bird. Matthew did not understand the Prussian's obsession with storms. He'd been like this all afternoon since the thunder started, and that had been a few hours ago.
There was another flash of lightning and the Canadian jumped. He didn't like storms at all. Sudden loud noises, sporadic bright flashes of light, and shrieking wind were not things Matthew found enjoyable. In fact, they scared him quite a bit. Which is why he was currently hiding out in their living room, one of the safest rooms in their house (why had he insisted on all these windows?), wishing to avoid the weather altogether.
He heard Gilbert's crazy laughter again as the loudest clap of thunder yet happened. He curled in on himself tighter. Storms were not ok. Could things get any worse?
Apparently they could, because with a particularly loud howl of wind, the power flickered out. Matthew was sitting alone in pitch darkness, which he also didn't like. He debated whether or not it was worth risking his life and protective chair to go in search of a flashlight or some candles. Gilbert was silent for a moment and Matthew wondered if the Prussian had finally come to his senses and realized storms were dangerous. Then he heard a quiet "kesesesese" from somewhere on his left.
The Canadian froze, listening carefully for Gilbert's footsteps. Too bad Gilbert had been working on his sneaking, wanting to be able to be as silent and undetectable as his boyfriend, and therefore could creep around without making a sound if he ever wanted to.
"Boo!" he yelled as he popped up behind Matthew's chair. Matthew gave a quiet startled yelp and jumped, limbs flailing from the tight position he had tucked them into.
"Kesesese! I got you!" Gilbert cried gleefully, throwing his arms around Matthew and pecking him lightly on the cheek.
Matthew couldn't form words; he was shaking too hard, his heart was beating too quickly. He was lucky he hadn't fallen out of the chair.
"Birdie? You ok? You're trembling. I didn't mean to scare you that badly...I'm sorry! Usually it doesn't freak you out this much. Was I just awesomely scary this time or something?" Matthew was fully aware of Gilbert's sneak attacks. Usually they involved momentary tickling or a kiss on the cheek, and the biggest reaction he'd have would be a violent twitch or a small noise. But this time was different; Matthew was already scared to begin with.
"I don't like storms." He responded, drawing his legs back up under his chin.
"But...they're awesome." Gilbert scooted him over gently, squeezing into the large armchair beside him and pulling Matthew into a comforting hug.
Another flash of lightning caused the Canadian to flinch violently while his Prussian boyfriend gave in to some sort of excited evil giggles. That's it. Gilbert must be possessed or something. He stopped his incessant 'kesesese-ing' when he noticed Matthew was less than thrilled.
"I brought you a flashlight!" Gilbert announced brightly, pulling it out of his sweatshirt pocket. Ok, so maybe he wasn't completely possessed. Matthew unstuck one of his tightly wound arms to take the light source.
"Thanks." he mumbled, clinging tightly to it.
"Of course! Anything for my amazingly awesome boyfriend!" Matthew blushed. He still wasn't used to Gilbert's sweet side. "And now I'm going to show you how awesome storms can be!"
Matthew frowned. "Gilbert..."
"Trust me, Birdie! I want you to like them too! And if anyone can convince you, it'll be the awesome me. Stay here until I get back, promise?" Matthew nodded warily. "Will you be ok by yourself?" Gilbert's excited tone evaporated into concern. The Canadian nodded again, thankful for the dim lighting to hide his blush. "Great! I'll be back in a few minutes!" and Gilbert ran off cackling to himself again. How he managed to navigate their house in complete darkness, Matthew would never understand. He was also slightly terrified about what Gilbert was planning. He just desperately wished he wouldn't scare him again, intentionally or otherwise.
"Birdie?" Gilbert called softly after Matthew had sat there for a few minutes, unmoving. "I'm back. I didn't want to scare you. I'm coming over to your chair now." Matthew kept the beam of light focused on Gilbert's chest.
Gilbert took his hands and untangled Matthew from his perch, turning off his flashlight and kissing his forehead reassuringly.
"Close your eyes." Gilbert said gently.
"Ok I see your point. Just close them when I tell you to, ok?" he pleaded.
Gilbert took his hand and led him down the halls of their house towards the sunroom. The room with the most windows. Matthew did not like this plan.
"Close your eyes."
Matthew really did not like this plan. At all. But he went along with it. Gilbert pulled him into their sunroom.
"Ok. Open them!"
Matthew's violet eyes blinked open and he gasped when they adjusted to the light. The room had dozens of little flashlights angled towards the windows, giving everything a soft white glow. The raindrops and water cascading down the windows on the roof reflected across the walls. In here, the thunder was more of an echoing rumble.
"Gil..." Matthew turned to look at him. He looked ethereal in this light. When the lightning came, it flashed in Gilbert's crimson eyes and his white hair and skin looked almost electric. He looked so alive, so energized. Matthew could make himself like storms just for the sight of Gilbert like this. Looking like he's having the time of his life, absolutely beautiful, staring at him with love shining through his eyes, perfect lips moving...Oh. Gilbert was saying something.
"Do you like it?" Gilbert repeated. He looked so delighted and hopeful, Matthew couldn't help but return his smile.
"Gil, I love it!" I love you. He'd wanted to say.
The Prussian grinned. "Dance with me?"
"There's no music."
Gilbert just shook his head and held out his arms. Matthew shuffled forward and put one hand on Gilbert's shoulder, the other interlacing their fingers together as Gilbert's other hand found its place on Matthew's back. They rested their foreheads together and began to sway as Gilbert quietly hummed a non-existent song.
"I would have taken you outside, it's more awesome that way, but I don't want you getting sick and I know you don't like getting wet." Matthew felt himself go pink. "But someday I want to kiss you in the rain. When it's not so windy or loud outside." The Canadian's blush deepened and he ducked his head slightly. "I've heard it's nice."
"It's not so loud anymore." Matthew whispered.
Gilbert's smile brightened. The storm had lightened considerably, finally resulting in a pleasant heavy rain. He carefully tilted Matthew's chin up and kissed him, earning a shy smile from the Canadian. Matthew stopped their gentle dancing and took a step away, keeping their hands together as he pulled Gilbert lightly towards the door.
Stepping outside, they were drenched instantly. Matthew pulled Gilbert along as he walked until he found a spot he deemed appropriate to stand. He turned to the Prussian with a smile, blonde hair clinging to his face. Gilbert gently brushed the strands away.
"I love you, Birdie!" He announced over the patter of the rain. Then with a hand on either side of Matthew's face, he brought their lips together as Matthew's arms wound around his shoulders. Lightning flashed in the distance and the responding thunder rumbled gently around them.
"Well?" Matthew asked after they broke apart, flushed and breathless. "As awesome as you expected?"
"No." Gilbert replied, equally breathless, still holding him close. "It was better!"
Matthew smiled. "I love you too, Gil."
Thanks for reading!
If anyone was wondering, I happen to love storms (until they get tornado-worthy).
I also flail like Matthew. Often.


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Erratic frequency and long routes have failed to enthuse students about the bus service launched by Delhi University in collaboration with the Delhi Transport Corporation from this academic year to help them navigate through the north campus easily.
"The frequency of the buses is low. One has to wait for a long time before getting a bus. And even if one is lucky then we have to wait for the bus to fill up before it starts moving," said Sweta Jagannathan, a first-year student of Hindu College.
Students said that it was better to just walk or take a cycle rickshaw because the buses often take long and circuitous way.
"It takes me a lot of time to reach the college since it has too many stops and it does not help when we are in a hurry to reach the college," said Sneha, a second-year student of St Stephen's College.
But then there are others like Karishma Kashyap, a second-year student of Ramjas College who is very happy with the bus service.
"It is really helpful. My transportation costs have come down drastically. It is too expensive to hire a rickshaw. Till last year they used to charge Rs 15 but they now demand Rs 30 till the metro station."
The bus service, which is available for the students and the staff of the university have a flat charge of Rs 5. The buses ply from Monday to Saturday from 8 am to 6 pm on three routes.
Meanwhile students at South Campus still have to manage without any special bus service. With even no private buses plying, students studying at the south campus or any of the four colleges, Ramlal Anand, Motilal Nehru, Atma Ram Santan Dharm and Venkateshwara situated at the Benito Juarez Marg have a hard time commuting.
"Inspite of writing to the DTC officials we have not received any positive feedback," said Dinesh Varshney, deputy dean, Students' Welfare, South Campus.