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Girls give support to Warrior's win
September 15, 2012
TAHOE CITY, Calif. – Everything about Thursday's Mount Rose League clash on Dollar Hill indicated that a rival was in town – from the soccer-hooligan-loud fans to the physically intense action on the field.
Competing in front of a screaming crowd of dedicated fans, the majority of them girls soccer players from both schools, the Whittell boys better capitalized on their scoring opportunities to earn a hard-fought, and sometimes contentious 3-1 win over the Lakers.
"These two teams are very evenly matched, and when you have a rivalry like this, it can get chippy at times," Whittell boys coach Royal Good said.
The girls, meanwhile, took part in two spirited competitions. After North Tahoe earned a 1-0 win in a closely contested game, both teams posted up in the bleachers and waged a boisterous rooting competition as the boys went to battle.
Waite scores two goals to lift Warriors
Play remained even between the North Tahoe and Whittell boys for much of the 80 minutes.
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About 28 minutes into the contest, Whittell's Palmer Chaplin crossed a ball to Mark Waite directly in front of the goal, where he and North Tahoe goalie Jose Chavez collided as they converged on it. Chavez took the brunt of the collision as the ball shot into the back of the net for a 1-0 Whittell lead.
The Lakers answered with an equalizer 2 minutes into the second half when Trevor Semmens sent a corner kick into a crowd in front of the goal and Riley McCarthy headed the ball past Whittell keeper Aubrey Felton, who was filling in for injured starter Austin Neil.
The home team did not have long to enjoy it. Just 2 minutes later, Waite burned a North Tahoe defender near midfield and drove all the way to about the 18-yard box, where he slowed up and blasted a kick into the upper-right corner of the goal, out of the reach of Chavez.
Nine minutes after that, Chaplin delivered a low corner kick to the near side of the goal, and a streaking Marshall Neil got a foot on it just ahead of the defending Lucas Early. Chavez, who made several nice saves in the game, set up the corner kick with a diving denial of a promising shot by Waite moments earlier.
The goal gave the Warriors a 3-1 advantage while noticeably dampening the Lakers' spirits. Austin Neil, who's nursing an injured hip, also returned to his goalie position for the majority of the second half, further limiting North Tahoe's chances for a comeback.
"We didn't play that bad. There were just times when they beat us to the ball and caught us flat-footed," said North Tahoe head coach Beto Alcaraz. "It looked like they wanted it more than us. They were getting to the ball first."
While Good said he was pleased with the way his team converted its opportunities, he conceded that the Lakers had just as many scoring chances in the game, perhaps more.
"It was a very good game," he said. "The first half we were a little sloppy. It wasn't as good of a first half as I wanted. But that first goal was huge. North Tahoe matched us pretty evenly. I give it up to them."
Whittell is now 9-1 overall and 1-1 in Mount Rose League play. North Tahoe is 4-5 overall and 0-2 in league.
Laker girls make first-half goal stand
The Whittell and North Tahoe girls would have played to a scoreless deadlock had it not been for a key play by a couple of substitutes in the first half.
About 25 minutes into the game, Sara Trimm dished off a pass to Audrey Johnson, who found some open space and slotted the ball past Whittell goalie Brittany Boulet for a 1-0 lead.
"We were kind of lucky to score that goal," said North Tahoe head coach Jon Rockwood. "Hats off to the subs who scored it. That was true sub fashion right there. They came in and were game-changers."
The Lakers might have added to their lead had it not been for the skilled goalie work of Boulet, who stopped several near goals in the game.
"Their goalie played a fantastic game," Rockwood said. "She was always well positioned. We took some great shots that she cut off the angle on."
Then again, the Warriors could easily have gotten on the board as well, but North Tahoe keeper Angela Wood played her own solid game in the goal to help boost the Lakers' confidence, Rockwood said.
"Angela, whether she knows it or not, carries this team," he said of his junior goalie. "She had three or four saves that were just great. Her reflexes are super fast. She came up big."
Whittell's most promising scoring opportunity came midway through the second half when Kenya Maltase had a breakaway run toward the goal. But North Tahoe sweeper Kami Abi-Nader used her speed to catch Maltase from behind and break up the play.
Shortly after, Maltase blasted a shot that Wood dove to save, while in the final 30 seconds of the game, Skylar Smith took a shot that sailed just left of the goal.
"Whittell played well. It was a very, very even game. It could have gone either way," Rockwood said. "It's always a fun game. We can't wait to play them again."
"It was a good game," Whittell head coach Patrick Kelly agreed. "North Tahoe has a really talented squad. They came out and played hard. It's always tough to play on the road in this league."
North Tahoe improved to 4-4-1 overall and 1-0-1 in Mount Rose League play, while Whittell fell to 8-2-1 overall and 0-1-1 in the MRL.

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BWW Reviews: MADAMA BUTTERFLY Hits Many of the Right Notes at the McCallum
I had the pleasure of attending the Teatro Lirico D'Europa MADAMA BUTTERFLY last evening at The McCallum Theatre and was once again swept away in Puccini's exquisite score and timeless love story. The vocalists were stellar; every one - everything else in the production was a little sub-par. Even though the singers were truly thrilling, their characters were cold and distant and it was difficult to find any empathy for them - except what was inherent in the score itself. But, even with its deficits, it was a wonderful way to spend an evening in the theatre.
Meliangee Perez's Cio-Cio-San (Butterfly) was vocally exquisite. She was definitively too old to play the young, fifteen-year-old geisha in both the maturity of her voice and her physique. Never once did she come off as the fragile Butterfly described repeatedly in the score and was never fully able to elicit true empathy for the tragedy of her circumstances. Antonio Pita's Pinkerton was also a pleasure to listen to, but his emotional range was limited and wooden. Their was no real connection between the lovers and thus no real emotional connection between characters and audience. The characters also came off as "middle-aged" and more cerebral than feeling.
Giorgio Lalov's direction was pedestrian and rather clumsy. This is one of the most passionate and heart-wrenching librettos in Opera and yet it was neither passionate nor heart-wrenching. It was all overplayed in a melodramatic style that, at times, was comic when it was meant to be dramatic. The blocking often seemed unnatural and stilted. And there were a couple of really "off the mark" choices made by Lalov. Pinkerton and Butterfly are only to have been apart for three years when she produces her son. The son was played by an obviously ten or eleven year old boy, almost as tall as Butterfly herself. When Pinkerton's American wife arrives with him in Act Three, they are there to take away Butterfly and Pinkerton's son - the stakes are dire. And yet Lalov has Kate blissfully waiting in the garden smelling the cherry blossoms with apparent joy - a contradiction to what she would be feeling under the circumstances. His direction not only did not support the piece, it may have been its emotional downfall.
The sets, the costumes, the lighting effects and the stage props were all of a community college level. The door on the Shouji kept getting stuck when Suzuki attempted to close it gingerly and at one point tea was served on a bed tray found at Walmart, totally out of period. My expectations were higher.
The supporting cast were equally tremendous vocalists. Dobromir Momekov's Sharpless was a solid anchor in the cast and Viara Zhelezova's Suzuki was more emotionally layered than her mistress, Butterfly. The trio between Pinkerton, Sharpless and Suzuki was one of the evening's finer moments. Perez's "Un Bel Di" aria was also a standout and was the only mid-show moment to receive spontaneous applause. Sadly, both Butterfly's suicide and Pinkerton's discovery of her body were cold, melodramatic and anti-climatic. The opera was performed with a full orchestra (bravo) and without any microphone amplification - which was, for the most part a big plus --- however, more often than not, the male vocalists were drowned out by the orchestra, especially in dramatic moments when both the singers and orchestra were at full throttle. The female vocalists all seemed to rise above the orchestra for most of the show.
Hooray for The McCallum for including Opera in their wonderfully eclectic season. Up next - the legendary Barbara Cook and then the incomparable Linda Eder. For more information or tickets visit www.mccallumtheatre.com.


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Injured pitchers pushing rehab forward
DENVER -- A Rockies rotation in desperate need of reinforcements looks closer to receiving exactly that, with Eddie Butler (right rotator cuff inflammation), Jordan Lyles (fractured left hand) and Tyler Chatwood (right flexor strain) all set to ramp up their rehab work.
After throwing a 35-pitch live batting practice session to Carlos Gonzalez on Saturday, Butler will take it easy for two days before tossing a 45-pitch simulated game Tuesday.
Lyles is set to throw 45 pitches Monday in his first bullpen session since fracturing the middle metacarpal on his left hand June 4. If all goes well, he's scheduled for a live, 30-pitch batting practice session on Friday.
Chatwood is set to throw live batting practice Wednesday for the first time since experiencing tightness in his right forearm in his previous live batting practice session last Wednesday. His pitch count for that next step has yet to be decided.
Brett Anderson (fractured left index finger) remains likely to return shortly after the All-Star break as he progresses on his rehab assignment as well. This flush of good news had manager Walt Weiss optimistic for the second half considering he's been scrambling for starting options since the calendar flipped to June.
"It looks like we're starting to get multiple guys back at the same time," Weiss said. "They're not coming back at exactly the same time but they're trickling in here. It will be good to get everybody back."
Christian Bergman (left fractured trapezium) will also participate in a live batting practice session with Butler on Tuesday. But he is ineligible to return from the disabled list until Aug. 20.
Cody Ulm is an associate reporter for MLB.com. This story was not subject to the approval of Major League Baseball or its clubs.


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The 2012 4-H State Shooting Sports Camp will be held at the Lake Cumberland 4-H Camp from April 4-7.
This is a statewide camp open to all youth in fourth through eighth grades.
Certified instructors have been recruited to present classes and range work.
Each camper will learn responsible, safe use of firearms and how to shoot in the six disciplines of the program: rifle, trap, archery, black powder, hunter challenge and pistol.
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Technology continues to revolutionize the way tasks are completed on the dairy, including your ability to get cows bred. One tool that has grown in use is ultrasound.
To better understand how veterinarians are using ultrasound to make effective reproductive decisions, the Dairy Cattle Reproduction Council asked two veterinarians about their experiences with the technology, and what the future holds for advancements in reproduction.


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Several local artists were honored at the 10th annual Fresh Art show at Summit Artspace at the opening of the exhibit March 7.
More than 50 works, ranging from paintings to collage to sculpture, were chosen for the juried exhibit, which encourages area artists to submit new works that present their chosen medium in a different way.
Four artists were awarded the Top Honors at the opening:
• BEST of SHOW award of $500 recognized Maggie Duff for her print, "Found Sign V."
• A new $200 award, the FRESH AIR award went to Nate Mayfield for his painting, "Stick a Fork in It."
• Second-place awards Andy Dreamingwolf of Brimfield, who received $150 for his painting, "Sister Ray."
• Third place for $150 went to Frances Lehnert of Kent for the pastel work, "Driving Miss Daisy."
In addition, five artists received an honorable mention:
• Eric Ford for his sculpture, "Untitled"
• Seth Madden for his painting, "LA Alhambra"
• Erin Miller of Kent for her mixed media work, "Effectiveness of Modern Medicine"
• Michael Swaisgood for his piece, "Pull"
• Sharon Wagner for her sculpture, "Migraine"
Alexandra Nicholis Coon, the director of the Massillon Museum of Art, was juror for this year's Fresh Art show.
"I thought this year's show was a wonderful mix of talent," Dreamingwolf said. "It introduced me to works by some artists whom I had never seen before and am looking forward to following. It's refreshing to see Akron's contemporary art scene flourish at shows like this."
Dreamingwolf said that his piece was part of a larger two-person show he has scheduled with artist Scot Phillips in 2015 in Cleveland.
"It's loosely based on Gus Van Sant's Drugstore Cowboy and an homage to old drugstores and five and dimes," he said. "It is a play on pharmaceuticals and novelties. The title 'Sister Ray' is taken from a Lou Reed penned Velvet Underground song in which Lou describes a scene of debauchery and decay. He said he envisioned Sister Ray as a transvestite smack dealer."
Miller, a textiles major at Kent State University who will be graduating this spring, said her work was "in response to a laparoscopic surgery I had in 2009 to remove an ovarian cyst."
"The surgery ended up causing significantly more damage than the cyst would have if it had been allowed to naturally and gradually reabsorb," she said. "This experience encouraged me to make more educated decisions about my health and reexamine the uneducated decisions I had made previously. I chose to weave the medical mask because it represents the cleanliness and sterility of the medical world. I wanted to represent the clean and dirty aspects of medicine so I chose a variety of materials as my weft."
Duff said she been collecting the signs used by "people who collect money near my house."
"I wanted to put myself in their shoes," she said.
The exhibit can be seen through April 19.
Regular gallery hours are Thursday from noon to 9 p.m., in conjunction with the Akron Art Museum, and Friday and Saturday from noon to 5 p.m.
Summa Health System provided the exhibition's cash awards.
Free workshops
Summit Artspace is presenting free workshops for artists to assist them in the development of their creative careers. All the workshops will be at Summit Artspace, are free and there is no advance registration.
• March 20 at 7 p.m., with photographer, Bradley Hart, Photographing your 2D & 3D Artwork for Reproduction
• April 8 at 7 p.m., sponsored by The Foundation Center, the Akron-Summit county library and Summit Artspace on Grant Seeking Basics for Individuals in the Arts.
Summit Artspace is at 140 E. Market St., one block east of the Akron Art Museum, and is a project of the Akron Area Arts Alliance, in cooperation with Summit County government. The building also houses individual artist studios and the galleries of Akron Society of Artists & Artists of Rubber City, - The Box. The Artists of Rubber City also is exhibiting its annual members show at the same time as the Fresh Art show.
In addition to regular gallery hours, the artist building is open during First Night and every Artwalk, the first Saturday of the month with open galleries, studios and when scheduled, performances by AAAA member organizations and area artists. The next Downtown Akron Artwalk will be April 5.
Email: email@example.com
Phone: 330-541-9438


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Experimental setup

Experimental setup

Revision as of 21:14, 26 October 2013
'What I cannot create, I do not understand'
This famous quote from Richard Feynman inspired our project. The initial idea of one physicist in our group was:
Let's build a cell!
So, of course, the biologists of our group tried to explain him, that a cell is far too complex and this is a crazy idea. If a biologist thinks of a cell, he sees an extremely complex structure, but the physicist just sees a simple black circle. So what is a cell, if you break it down to a simpler level?
A compartment separating a reactive center from the outside, combined with controlled means of transport in and out and some modifications on the outside. An intelligent, microscopically small reactor. And our idea of a Smart Nanoreactor was born.
First of all, we needed a compartment. In cells, the compartment is made out of a phospholipid bilayer membrane that forms the plasma membrane. But this being already nature's strategy, we wanted to try a different thing and decided to use block co-polymers to build the nanoreactor wall.
What are polymers?
Polymers consist of structural or repeating units of low molecular weight covalently connected to each other to give high molecular weight compounds. A polymer that consists of two different repeating units is referred to as a co-polymer (Odian, 1981). The covalent linkage of two intrinsically different homo polymers leads to the formation of block–copolymers. Block-copolymers consist of at least two, covalently bound, segments or blocks of different Homo polymers which lead to the formation of di-block or tri-block copolymers. For instance, a tri-block copolymer can have a general form An-Bm-Cp, with A, B, C, being different monomer types constituting the different blocks. The subscripts n, m, and p stand for the degree of polymerization, i.e. the average number of each monomer present in each respective block (McGrath, 1981).
Why using polymers for our Nanoreactor?
There is growing interest in polymersomes due to their potential application in biotechnology and pharmacology (Blanazs et al., 2009) Currently, a great potential lies in their ability to mimic biological membranes by establishing aqueous three dimensional functional cavities. The stability of these membranes is higher than those of biological lipid-based vesicles. Thus, polymersomes consisting of amphiphilic block copolymers hold promise as alternative vesicles in biotechnological and biomedical applications (van Dongen et al., 2011).
Two important parameters of the membranes are their permeability and their stability. The advantage of synthetic block-copolymers as the building blocks of these nanoreactors is their higher stability over lipids due to the increased length, conformational freedom, and slower dynamics of the underlying polymers.
Other desired characteristics for the Nanoreactor the polymers can achieve are:
- The shell has a hydrophilic, biocompatible low-protein-binding surface.
- The shell can act as a bio membrane for protein reconstitution.
- The nanovesicle can encapsulate hydrophilic substances.
- The shell material has enormous possibilities for molecular functionalization.
The nanovesicles can thus be employed as specific targeted drug delivery systems (e.g. biotinylated vesicles), stimuli responsive nanocontainers (e.g. pH sensitive) or nanoreactors.
Towards the above goal, two polymers, an ABA tri-block polymer and an AB di-block polymer were investigated.
Tri-block polymer
This ABA tri-block copolymer consists of a middle block of PDMS (poly (dimethyl siloxane)) and two PMOXA (poly (2-methyloxazoline)) side chains. These copolymers self-assemble in aqueous solution to form highly stable, closed vesicles with a controlled diameter of typically less than 200 nm.
Tri-block polymersomes were prepared by film rehydration using PBS buffer (pH 7.4) as rehydrating solvent. The polymersomes were observed using cryo-SEM and cryo-TEM, and the particle size was characterized by DLS (Dynamic light scattering).
Cryo-TEM images clearly depict lamellar and large vesicle formation, which is further confirmed by DLS showing an average diameter of ~950nm and a broad particle size distribution. These are not as desired, so these polymersomes were further subjected to an extrusion process using Ultra-filtration equipment.
After extrusion, the samples were observed under cryo-TEM again and their size distribution was measured by DLS. Ultra-filtration process extruded larger vesicles and lamellar structure which is well seen in TEM images. In the DLS measurement, the peak of diameter ranging from 226 to 238 nm is better defined and corresponds to 50% of the total polymersomes.
Di-block polymer
We were also interested in thermo-responsive block copolymer, poly(2-cinnamoylethyl methacrylate)-poly(N-isopropylacryl amide), pH-sensitive and photo-cross-linkable block copolymer. They can function as on/off switches depending on the external pH stimulus due to the pH sensitivity of their cross-linked membrane.
The block copolymers undergo spontaneous self-assembly into the desired polymersome structures by raising the pH of the aqueous solution from 3 to 10. To preserve the vesicular shape of the polymersomes under varying conditions, cross-linking of their membranes is highly desired.
These di-block polymers self-assemble into polymersomes of uniform size of 100-120 nm and later expand till 140 nm as the pH is lowered from alkaline condition to acidic state. Thus, they act as pH responsive polymersomes which can tune its release of biological entities based on environmental pH condition. Such property makes them demandable in field of drug therapy specifically in treating cancer.
The formation of the polymersomes was analysed using cryo-TEM and the particle size distribution was measured with DLS. In this case, the distribution is very uniform with a peak at 140 nm.
The pH responsive nature of di-block polymer was investigated by the release profile of Doxorubicin, an anti-cancerogenic agent which showed slow release at pH 7.4 and faster release at pH 5. This is a proof that the polymersomes were effectively cross-linked by UV.
DNA Origami channel
To allow transport inside and outside the smart nanoreactor, a channel is needed. Instead of using a protein channel, we decided to use a DNA origami channel, because it is more robust, and we can easily design it according to the characteristics of the polymer wall.
What is DNA Origami?
In DNA origami a circular ssDNA molecule of several thousand base pairs, called scaffold, is folded with the help of short ssDNA strands, the staple strands. The staple strands bring together two different zones of the scaffold, and by varying their sequences different 2D or 3D shapes can be achieved.
DNA origami has unique properties, such as an addressable surface, which enables selective functionalization with biomolecules and nanomaterials. The origami can also be combined with top-down nanotechnology, such as placement on a fabricated substrate. The technology can also be used in single-molecule imaging, where FRET pairs or fluorophores can be constructed on designed DNA origami structures. Furthermore, DNA origami can be easily combined with other DNA nanodevices such as DNAzymes, DNA beacons or DNA walkers acting on DNA origami. Hence, DNA origami technology has practical potential in various research fields.
The origami channel was designed to be properly integrated into the polymersome wall. For this, the dimensions were restricted to the thickness of the wall, as well as to the length of the scaffold. Furthermore, the dimensions of the porus are determined by the dimensions of the substrate and the enzyme: it has to be big enough so the substrate and products of the reaction can easily go through, but small enough so the enzyme stays encapsulated inside the polymersome. The final dimensions are the following:
- Length: 34 nm
- Diameter: 25 nm
- Pore diameter: 6 nm
Scaffold and staple types
For the design, the caDNAno program was used (see http://cadnano.org/), and the stability of the structure was tested with CanDo (see http://cando-dna-origami.org/).
The scaffold used in this design is 7560 bp long, derived from the M13p18 Escherichia coli virus. Its sequence was taken from the cadnano design program. Bending it into a structure with a 78 helix bundles, the desired length is achieved. The channel contains four types of staples:
- core: 118 staples give stability to the whole structure.
- edge: 39 staples give stability to the edges.
- helpers: 12 staples have a 5' protruding end with 5 adenines, which should help the anchors point to the right directions.
- anchors: 12 staples have a 3' protruding end of 21 nucleotides (7 nm) which will allow the binding of the oligonucleotides that carry the hydrophobic modifications. Between the corresponding complementary sequence to the scaffold and the protrusion, 5 thymine nucleotides serve as spacer and hybridize with the 5 adenines present in the helpers.
- no_hang: can be used instead of the anchors to generate a channel without any anchors sticking out of the channel walls. They can serve as a negative control when introducing the hydrophobically modified oligonucleotides.
Their sequences and also the map of their distribution in the scaffold can be donwnloaded from the lab book.
Folding and characterization
The mixture of scaffold and different staples was subjected to a thermal annealing ramp, that allowed the folding. In an initial experiment, different concentrations of Magnesium chloride in the folding buffer were tested, and the resulting structures were analized by atomic force microscopy (AFM) and electrophoresis to determine the best concentration. The range 10 to 14 mM MgCl2 were the best, so we chose the 12 mM concentration for further experiments.
In the AFM images the height and width were measured, obtaining the approximate values of 7 and 60 nm respectively. The height appears to be much lower than the expected, due to the high absorption of the channel to the substrate - mica - used for imaging. Mica is negatively charged, and so the Magnesium chloride in the samples interacts with the surface creating a positively charged substrate. Thus, the DNA origami channel can interact tightly with the surface, modifying its structure. The width appears much bigger, which can be due to the tip-sample convolution.
TEM analysis
The 3D structure was then corroborated using TEM and cryo-TEM.
For the TEM images, the diameter and length of 20 different structures were measured. In this case, the dimensions of the channel and also its shape correspond accurately to the design.
Hydrophobic modifications
For the channel to be incorporated inside the polymersome membrane spontaneously, a hydrophobic modification was introduced to a short oligonucleotide that hybridizes with the anchors. Two different types of hydrophobic modifications were performed.
5' Palmitate and arachidic acid modification
An amino group was introduced to the 5' end of the oligonucleotides, that afterwards reacts with the carboxylic group of the molecule containing the hydrophobic fatty acids palmitate and arachidate. The reaction used is an N-acylation, which requires some activations. More details of the reaction and reagents used can be found in the Lab book.
The modified oligos were purified with HPLC and the purity of the product was analized by mass spectrometry.
3' Cholesterol modification
Oligonucleotides for 3' cholesterol modification were synthesized on a solid support already carrying the cholesterol molecules. In this case the sequence was shorter for the modification to be slightly separated from the walls of the channel. The modified oligos were purified with HPLC and the purity of the product was analized with mass spectrometry.
Incorporation of the hydrophobically-modified oligonucleotides into the origami channel
The success of the incorporation of the hydrophobically modified oligos into the DNA origami channel was analyzed by electrophoresis.The DNA origami channel was folded with the presence of no_hang staples, so a structure with no anchors was produced. This channel without anchors was put into contact with the oligonucleotide complementary to the anchors without modification, and also with the oligonucleotide with two different types of hydrophobic modifications. This served as a negative control as no difference in the electrophoretic mobility should be observed.
In parallel, the DNA origami channel with the anchor staples was produced in the present of the oligonucleotide complementary to them without the hydrophobic modifications, and the same oligonucleotide with two different types of modifications.
In the electrophoresis gel a shift can be observed between the electrophoresis mobility of the channels which don't contain the anchors and the ones which contain them. This is because the shape of the structure is affected by these sticking out DNA strands in a way that they cannot run as fast in the gel. Moreover, when introducing the oligonucleotides modified with 5' palmitic and arachidic acid that hybridize with the anchors, a further shift is observed, indicating that this modification is correctly incorporated into the channel. As for the 3' cholesterol modification, no shift is observed, but this can be due to the small volume of the cholesterol molecules in respect to the channel and their position being closer to the channel walls. In both modifications, though, a remaining of the sample inside the walls can indicate a certain level of formation of big complexes unable to enter the gel due to aggregation of the channel through their hydrophobic regions, specially in the 5' palmitic and arachidic acid modified channel. This is a good indicator that the hydrophobic modifications were properly incorporated onto the DNA origami channel.
Internal reaction
The aim of our project is to create a nanoreactor. Inspired by how cell metabolism is organized in specific cellular compartments, we decided to design a system which is capable of hosting enzymatic reactions. Inner compartmentalization allows fragile processes to remain protected against undesired influences, such as proteolytic or microbial degradation and also one could expect enhanced reaction probabilities and efficiencies due to spatial confinement or immobilization of enzymes.
Choice of reaction
There are several requirements for the enzyme which can be entrapped. The enzymes should be large enough so that they are not able to escape, especially if channels or pores are formed in the system. Another requirement is that the reaction is easy to detect. Examples include enzymes which are able to produce fluorescent or chromogenic products, such as myoglobin, Candida Antarctica lipase B, glucose oxides, horse radish peroxidase, urease and α-chymotrypsin.
The protein of our choice was collagenase. Collagenases are endopeptidases that degrade the helical regions in native collagen preferentially at the Y-Gly bond in the sequence Pro-Y-Gly-Pro, where Y is most frequently a neutral amino acid. This protein is very abundant in many species and it is easy to obtain from the bacteria Clostridium hystoliticum. The isolated enzyme is composed of 7 different proteases ranging in molecular weight from 68-130 kDa, thus the protein is big enough and there is no possibility to pass through the channel. Furthermore, the reaction can be detected easily with spectrophotometric assay.
Fluorescent labeling
The protein was conjugated to amine-reactive fluorescent dye in order to prove its entrapment inside the polymersomes. TAMRA (Tetramethylrhodamine) NHS was used to perform the fluorescent labeling. TAMRA belongs to the group of the long-wavelength rhodamines with excitation maximum at 555nm and emission maximum at 580 nm.
The degree of labelling with TAMRA dye was estimated to be 18 dye molecules per enzyme.
Enzyme reaction assay
The enzymatic assay for determining the rate of the reaction was carried out using the peptide FALGPA, standing for N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala. There is sufficient structural similarity between the 2-furanacryoyl group and Pro for FALGPA to be an excellent substrate for collagenase.
The molecular weight of the substrate is 476 Da. The maximum molecular weight of particles that can go through the polymer walls is less than 400 Da. Thus, the substrate will not be able to penetrate the polymer walls, but only enter the nanoreactor through the channel. Furthermore, the substrate being uncharged, the negative charge of the DNA won't interfere with its transport.
This peptide has absorbance at 345 nm and the reaction is monitored through continuous spectrophotometric measurement at this wavelength. The assay is possible because of the blue shift in the near-ultraviolet absorption band of the furanacryloyl peptide when the peptide bond between the first and second residues is hydrolyzed. This means that after cleavage of the peptide, the absorbance at 345nm starts to decrease. The enzyme activity before and after conjugation to the dye is characterized through this assay.
The conjugation to the fluorescent dye leads to a slight decrease in the activity of the enzyme. The FALGPA units are calculated, taking into account the slope of the linear fit. The unmodified enzyme is estimated to have 3 FALGPA units/ml and the conjugated one – 2.6 FALGPA units/ml.
Since the polymersomes can cause light scattering events, UPLC measurement of the reaction products was applied for better accuracy of our experimental results.
Smart nanoreactor
Incorporation of the channel
The DNA origami channel was incorporated during the polymersome preparation and was characterized using TEM and Cryo-TEM.
For the tri-block copolymer, the polymersome formation was not affected by the addition of the DNA origami channel, and the DLS analysis confirmed that the polymersome size distribution was the same. In some of the polymersomes, particles across the membrane were observed. This indicates that the origami channels were successfully incorporated.
To further confirm the incorporation of the channel in the wall, gold nanoparticles can be attached to the DNA channel. This will make them more distinguishable in the TEM and cryo-TEM analysis. This is planned as a follow-up experiment.
As a further confirmation of the channel incorporation into the polymer wall, and its functionality, we performed a dye release assay.
For the di-block copolymer, the polymersome formation seemed to be affected by the presence of the DNA origami channel. The polymersome surface became rough and its stability decreased. The polymersomes had a smaller diameter than previously observed and were present in a smaller amount. This made the observation of the DNA channel incorporporation difficult.
Dye release assay
In order to confirm the functionality of the origami channels and their successful integration into the polymersome walls, a dye release experiment was performed. The fluorescein dye was encapsulated into the polymersomes in presence and absence of the channels. Fluorescein molecular weight is 332.29 Da and it can pass through the polymer walls, although with some difficulties. The incorporation of the channel, though, should facilitate the exit of the dye. All the samples were placed inside dialysis bags and the fluorescein concentration outside the polymersomes was measured by UV/vis spectroscopy.
As a positive control, the release of fluorescein from the dialysis bag without polymersomes was also performed.
Tri-block polymersomes show around 80% of release within 24h whereas tri-block polymersomes with DNA channels exhibit complete release of dye. Because they allow the fluorescein to go through them, this is a strong hint of the successful incorporation and functionality of the DNA channels.
Di-block polymersomes with and without origami channels have similar release profile. The release of dye from the di-block polymersome was less than 50% which proves association of dye with polymer. It is in agreement with confocal LSM images presenting strong adhesion of the dye molecules with di-block polymer which may inhibit polymersome formation. In this case, thus, the dye-release assay cannot be used to prove the incorporation of the channels due to the interaction of the dye with the polymersome walls.
After 24h, the samples were also imaged by C-LSM (Confocal Laser Scanning Microscopy).
Tri-block polymersomes without channels clearly trapped the dye molecules which could be observed in C-LSM images, whereas the polymersome with DNA channels released most of the dye and residual polymers containing dye could only be imaged with 10x higher laser power and increased gain. This shows that in the latter case, almost all the dye could go outside of the polymersomes.
Di-block polymersomes were highly destabilized in presence of dye. Only a few polymersomes encapsulated dye molecules similar to tri-block polymersome. Most polymersomes were in disrupted state and strongly adhering fluorescence dye molecules. This result is in agreement with cryo-TEM images where we observed the similar structure of polymersomes.
Encapsulation of the enzyme
The collagenase conjugated with the TAMRA fluorescent dye was encapsulated in both polymersomes. Dialysis was performed to eliminate the non-encapsulated enzyme. The encapsulation efficiency was calculated as the concentration after dialysis divided by the concentration before dialysis. For the Tri-block polymersomes, it was of around 70%, whereas for Di-block polymersomes, it resulted to be of around 30%.
The samples were then imaged by C-LSM. Particles that correspond to the polymersomes with the encapsulated enzyme could be observed in both Tri-block and Di-block polymers.
In order to prove that after dialysis all the free enzyme is removed, the polymersomes were incubated with substrate solution and UPLC measurements were taken at different intervals of time. Even after 16 h of incubation, no products were detected, meaning that no free enzyme is present in the solution and that the enzyme is successfully encapsulated inside the polymersomes.
Smart nanoreactor synthesis
Until now, we demonstrated that both DNA channel incorporation and enzyme encapsulation were successful. The assembly of DNA channels and encapsulation of active enzyme at the same time are now to be demonstrated.
Tri-block polymer
In presence of enzyme, the DNA channel incorporation into membrane of polymersome was found to be successful as shown in the cryo-TEM images. We hypothesize the integration of DNA channels into the polymersome membrane from image contrast modulations around the polymersome membrane which are definitely not present in absence of DNA channel or Enzyme. In addition, the channels were also found to be perpendicular attached to the polymersome surface which may or may not be associated with polymersomes. Further, confirmatory results can be obtained by three dimensional cryo-TEM image collection and reconstruction.
In order to obtain further evidence for DNA channel incorporation into polymersomes, negatively stained samples were inspected under TEM. One should consider, though, that the polymersomes get at least partly collapsed during the vacuum procedure necessary for the TEM analysis. In absence of polymer, the DNA channels are clearly visible as cylindrical motifs with sharp defined contours. While in presence of polymer, the DNA channels are embedded in a diffuse matrix, potentially a polymer shell, which is attached to the channel.
In order to confirm our hypothesis, the enzymatic assay was performed and samples taken at different time points were analyzed using UPLC.
Di-block polymer
The di-block polymersomes had a highly distorted membrane instead of intact polymersomes, in the same way as it was observed with only channel incorporation.
Smart Nanoreactor assay
The enzymatic assay was performed to prove that our system works: the channel allows the transport of the substrate in and out of the polymersomes, the encapsulated enzyme is still active and the reaction takes place.
The reaction assay was performed after dialysis of the samples, so that no free enzyme is present in the solution. For the samples with DNA channel, two types of reaction were set, one with higher concentration and another with lower concentration of the polymersomes, to distinguish which one is the best to observe the reaction. The reaction was performed and samples were taken at 3h, 8h30min and 48h. Those were frozen and the reaction products were analyzed by UPLC.
For the tri-block polymersomes, conversion of substrate into product could already be observed after 3h incubation, even in the samples with lower concentration. However, the reaction rate in these samples is slower because even after 48h the majority of the substrate remained unconverted. Nevertheless, the reaction for the tri-block polymersomes could also be observed in the sample without channels. For this reason, this assay should be repeated with an optimization of the enzyme concentration and the dialysis time.
Previous reactions with the polymersomes without channels and the encapsulated enzyme showed that there was no conversion of the substrate, so in this case the apparition of the product could be due to a too short dyalisis time. As a follow-up experiment, proteases will be added outside the polymersomes before the reaction assay in order to make sure that no free enzyme is present in the solution, thus proving that the enzyme inside the nanoreactors is protected and still active.
In the case of di-block polymersomes, the samples showed no reaction, even with higher concentration of polymersomes. This correlates with the previous results that showed that the formation of these polymersomes was affected by the incorporation of the channel, and so no enzyme could be incorporated.
Future work
Our project does not finish here. There is a set of planned experiments that due to the time constraint was impossible to perform. The ones that catch our interest are the following.
Quantifying the incorporation of the channels
We will attach gold nanoparticles of around 2-5 nm in size that have linked to their surface a ssDNA sequence that can hybridize with the scaffold of the DNA origami channel.
This will help distinguish the DNA origami channel in the TEM and cryo-TEM imaging, and so a quantification of the channels that are present in each polymersome will be made.
Structural studies of the origami channel
In collaboration with Prof. Dr. Rasmus Schroeder, in Heidelberg University, we will do structural studies of the DNA origami 3D structure in the nanometer scale.
Optimization of the Smart Nanoreactor
The formation of the Smart Nanoreactor will be optimized with regard to the concentration of enzyme, DNA channel and coblock polymers.
Furthermore, reaction could be observed in the negative control. Previously to the reaction assay, proteases will be introduced outside the polymersomes that can degrade the enzymes that are not incorporated. Thus, we will prove that the observed products are due only to the encapsulated enzyme.
In the case of the di-block polymersomes, their formation with the presence of the origami channel was affected. Thus, we will further investigate their formation with lower concentrations of the origami channel.
Antibody immobilization
For targeted delivery in the in-vivo studies, the coblock polymers will be functionalized with a suitable linker for the antibody attachment. Thus, we will be able to specifically target cells with our Smart Nanoreactor.
In-vitro and in-vivo studies
In-vitro studies with cell cultures will be performed to test the delivery of the antibody-modified nanoreactors to the desired cells.
Furthermore, in collaboration with Prof. Dr. Michael Brand in CRTD, we will perform in-vivo studies using Zebrafish, because due to their transparent body, we will be able to easily trace the Smart Nanocontainer and analyze its efficiency in targeting.
Drug delivery system
In recent years DNA origami reached incredible popularity in nanotechnology due to its vast design possibilities and its broad potential applications. While having numerous advantages it is still exceptionally expensive to produce in high amounts for mass applications, whereas inorganic polymers, another range of materials available for biomolecular design can compensate for this disadvantage. Nowadays they can be found in almost every aspect of our lives, in medicine and technology through their wide range of different functionalities and their moderate price. In our project we aim to combine those two artificial materials into one device: functional and responsive polymers as carrier material with highly flexible designable DNA origami for more complex access points to gain the best of both of them.
In the main application, presented in our video, we describe our Smart Nanoreactor as a new form of drug delivery system, which takes great advantages of the polymersome's role as carrier for multiple different other functional components. In chemotherapy a big range of treatment methods use drugs which are damaging both cancerous as well as healthy tissue, putting the patient in a high stress during the treatment. But with our Smart Nanoreactor we offer a solution for this problem. As a first step the Nanoreactor is injected into the patient and through antibodies immobilized on the outside of the sphere, the device is targeting specific pathogenic tissue like cancer and is localized only in this area. In the second step an inactive form of a pro-drug gets systematically introduced into the patient, so it is localized in healthy as well as pathogenic tissue. Now the pro-drug can enter the Nanoreactor through the origami channels and the encapsulated enzyme can convert the inactive pro-drug to its active state in which it is damaging both healthy and pathogenic tissue. After exiting the Nanoreactor again the active drug can interact with its surroundings, due to the location of the Nanoreactor the closest cells are the cancerous tissue. After the targeted cells are treated the immobilized antibodies loose their binding partner, the reactor disattaches and is filtered out of the body through the kidneys.
pH-responsive Nanocontainer
Another powerful feature of our device is the use of a pH-responsive polymer, which enables us to add even more functionality to our Nanoreactor. The described di-block copolymer has tight and stable walls in basic pH but gets leaky in acidic milieu. We could use this to create another route of transport for molecules that are too big to fit through the origami channel. In high pH the container circles through its environment and takes up small molecules from the outside, an enclosed enzyme crosslinks them together until they are too big to exit again through the channel. After the container reaches surroundings with lower pH, its walls become more porous and allow the exit of its cargo through pores in the polymersome. A great advantage is the reversibility of this process, so when the vesicle is empty, it can be brought back to basic pH, the walls are sealed tight again and the reactor can take up more substrate through the DNA channel.
This feature could not only open a variety of applications in medicine where our device can react to physiological changes in pH but also in technology and environmental engineering for example. Synthesizing processes can be automated and our Nanoreactor can respond to different stages of a pH changing reaction or it could be used to clean freshwater supplies via independent sampling of contaminated wells.
Smart Nanofactory
The possibilities of our device for technological applications do not end here. Observing nature and discovering the formation of polymersomes inside bigger polymersomes during Cryo-TEM analysis, another great range of technical applications came to our minds. If we extend this process to creating complete Smart Nanoreactors encapsulated in other Nanoreactors we can create whole Nanofactories in the future, similar to the different lipid vesicle compartments in living cells like vacuoles.
Combining different reactors with various different DNA origami channels, polymersomes and enzymes we are able to design a system, which can independently catalyze complex chains of reactions. Immobilized targeting proteins on the outside of polymersomes and their sizes act for sorting of the reactors, while the different channels determine the order of entry that the substrates have to use to complete the full chain of a reaction. Besides easy separation of product molecules from enzymes, our device has the advantage of formation of big structures by different enzymes in a complex order, one can even create repeats and loops by incorporating a range or Nanoreactors within each other, all of it independently.
This was just a small trip through the wide spectrum of applications for our Smart Nanoreactor, but there is far more possible in the future. And all of this just with a plastic ball.
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NEW YORK—What we learned Saturday night while watching the Louisville Cardinals complete a stunning second-half comeback and earn their second consecutive Big East Tournament championship with a 78-61 victory against Syracuse:
Never count out the Cards
At one point in the second half, Syracuse led 45-29. Let that sink in for a moment. In the second half of a game they lost by 17 points, the Orange led by 16 points with less than 16 minutes remaining in the game.
This Louisville defense is absolutely good enough to win a national championship. That's not a new revelation, by any means, but this second half is the section of video that will keep opposing NCAA Tournament coaches up nights, the video that will wake them up in cold sweats if they're able to fall asleep.
This was a Big East Tournament championship the Cardinals will remember long after they've joined the ACC, and it's one Syracuse won't forget, no matter how soon they jump leagues.
MORE: Bubble Watch | Automatic bid winners | Countdown to Selection Sunday | Printable bracket
In the first half, Louisville shot 25.9 percent and scored 22 points. In the second half, the Cardinals shot 53.3 percent and scored 56 points. The difference, Louisville coach Rick Pitino told a crowd of reporters in a hallway deep inside Madison Square Garden, was shot selection and patience.
"It taught us a great lesson tonight," he said. "To really rush your offense like that in a big-time game taught us a lesson. I told them at halftime, I said, we're going to go out and play like Louisville in the second half. We know what comebacks are all about. We're going to get into our press because we're going to take good shots."
This was a Big East Tournament championship the Cardinals will remember long after they've joined the ACC in July 2014, and it's one Syracuse won't forget, no matter how soon they jump leagues (for the record, that happens on July 1 this year).
"For me, it's the end of an era," Pitino said. "The Big East comes to an end, and to be back-to-back champions, to have a captain be back-to-back MVPs (Peyton Siva), it's as special as it gets. And to have it happen in Madison Square Garden is a memory of a lifetime."
Nets not worthy
This time around, the Cardinals didn't bother to cut down the nets, as is tradition for team that wins a conference tournament. They have their sights set higher than "just" the Big East Tournament.
"We have a goal in mind," Siva said. "We want to savor the moment. God-willing, we make it to the national championship, but we've got to take that one game at a time."
It's an odd move, but a confident one. And it's an especially stark contrast to what happened at Indiana, when the Hoosiers cut down the nets to celebrate a share of the Big Ten regular-season championship even after losing at home to Ohio State.
The Cardinals hope to only cut down the nets once in postseason, in Atlanta after winning the national championship.
"Let's see if we can do something special," Pitino said. "And if it happens for us, it happens. If not, we didn't miss it too much because we cut it last year."
Harrell a beast
Freshman power forward Montrezl Harrell, who was originally committed to Virginia Tech but opted to attend Louisville after Hokies coach Seth Greenberg was fired, played the game of his life Saturday night.
He had seen relatively limited minutes down the stretch—not more than 19 since Jan. 9—but was absolutely instrumental in Louisville's comeback. His length (he's 6-8 with a 7-6 wingspan) was a big part of the game plan.
"You have to beat Syracuse's zone by going high to low, or by going diagonally from high to corner," Pitino said. "You can't beat it by passing to the wing."
His massive dunk with 9:51 left in the second half, off an excellent dish from point guard Peyton Siva, gave Louisville its first lead of the game. That wasn't Harrell's only big play, though. There's a reason he stayed on the court for 24 minutes, after playing just eight on Friday night against Notre Dame.
He attacked the rim relentlessly. He rebounded. He blocked a huge shot with under four minutes left in the game. Heck, he even knocked down a jumpshot.
"Coach didn't tell me it was gonna be a big game for me, he didn't say he's gonna need me, he didn't say none of that," Harrell said. "He just said that everybody as a team, we had to play hard."
Harrell definitely played hard. He scored 20 points—his previous career high was 14 vs. Missouri-Kansas City in December—and grabbed seven rebounds and was the physical presence the Cardinals needed to complement center Gorgui Dieng.
MCW is special
There was a time not so long ago that Syracuse point guard Michael Carter-Williams was considered a no-brainer All-American. Syracuse's stumble to the end of the regular season—MCW's struggles were only part of the slide—ended that conversation, but the talented sophomore reminded us this week at Madison Square Garden why he's still regarded so highly by NBA scouts—draftexpress.com has Carter-Williams going 13th overall in its mock draft.
He finished with 11 points, nine assists and four turnovers.
In the first three games of the Big East Tournament, he handed out 27 assists and came up with seven steals. In the first half Saturday against Louisville, MCW delivered two of the biggest field goals to that point of the game. The Cardinals had cut Syracuse's eight-point lead down to four points on a 3-pointer by Kevin Ware with 5:25 left in the first half when MCW took over two possessions.
On the first one, he drove to his left, fending off a Louisville defender draped all over him—that's the only way Louisville defenders know how to defend—and converted to make the score 24-18. On Syracuse's next possession, he again attacked the rim, and somehow, while falling down after hard contact by Chane Behanan, flipped up a shot with his left hand that banked in to give the Orange again an eight-point advantage. Those two baskets started a 13-4 run for Syracuse to close the first half and give the Orange a substantial 35-22 halftime lead. Not nearly substantial enough, it turned out.
And in the second half MCW again struggled. He wasn't the only one who couldn't handle Louisville's second-half pressure, but as the primary ballhandler, he's probably most responsible for SU's dozen turnovers after the break.


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Brandon Meriweather intends to go low, tear people's ACLs and end careers
Redskins safety Brandon Meriweather served his one-game suspension (reduced from two games on appeal) Sunday, missing a loss in Denver. He will be back on the field in Week 9 against San Diego and, apparently, offensive players everywhere ought to be on the lookout.
Speaking with the media Monday, Meriweather said that if the NFL is going to punish him for hitting high, he'll have to target another area of the body.
"To be honest, you've just got to go low now," Meriweather said, according to ESPN 980's Chris Russell. "You gotta end people's careers.
"You gotta tear people's ACLs," Meriweather continued. "Mess up people's knees. You can't him them high anymore. You've just got to go low."
Perhaps not the smartest comments for a player whose rather reckless play already has landed him squarely on the NFL's radar. Meriweather drew his suspension after delivering a pair of penalized hits in a loss to Chicago, one on Alshon Jeffery and another on Brandon Marshall. Earlier this season, Meriweather knocked Packers running back Eddie Lacy from a game with a helmet-to-helmet hit and he's been fined in the past for his on-field actions.
Marshall took particular exception to his run-in with Meriweather, calling on the NFL to punish Washington's safety severely: "I respect the league trying to better our game and guys like that, maybe he needs to get suspended or taken out of the game completely. ...
"Guys like that just don't understand. Those are the guys that are in trouble. They really don't have anything to do after football because they think it is all about football."
Marshall told ESPN's Linda Cohn on Listen Closely that Meriweather reached out to him after those comments, seeking some explanation. "I talked to Brandon ... I just tried to express to him how I felt -- that one, I don't understand why he would hurt himself physically and financially; and two, I just explained to him that we've got to accept the game for what it is. When you look at his history, there's a pattern there."
Apparently, whatever Marshall said to Meriweather did not land on friendly ears. Meriweather snapped back at Marshall's initial comments on Monday, per Zac Boyer of The Washington Times.
"He feels like I need to be kicked out of the league? I feel like people who beat their girlfriends should be kicked out, too," Meriweather said, referencing a civil suit that a former girlfriend filed against Marshall, alleging domestic abuse. That suit was thrown out and Marshall's name cleared.
Meriweather may not have all the information on that case, because he added: "You tell me who you'd rather have: Somebody who plays aggressive on the field, or somebody who beat up their girlfriend?"
The one-game suspension cost Meriweather approximately $75,000 in salary, on top of the $42K he was fined for his hit on Lacy. The NFL also assessed Meriweather a $75,000 fine in 2010 because of two hits on then Baltimore TE Todd Heap, and he racked up $45,000 in additional penalties the following season.
Meriweather's recent suspension was the most severe punishment he has received thus far, though his argument that he has no choice but to hit low is one brought to the table by multiple defenders in the past. (Meriweather took it a step further than others with his comments about ending careers and tearing ACLs.)
Houston safety D.J. Swearinger took a similar position in the preseason, amid criticism lobbed his way after he ended Dustin Keller's season with a hit to the knee. Baltimore's Chris Canty later came to the defense of his team's safety, Matt Elam, after Elam injured Randall Cobb with a similar blow. "It's a legal hit, it's within the rules," Canty said of Elam's tackle.


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The Kurzweil K2000 is a classic synthesizer/sampler workstation. Everyone I know who ever owned one still has it in their studio hooked up and running. Every few months I hear someone and talk about it's magic sound and abilities. One interesting tidbit you may not know is that the synth division of Kurzweil is owned by a Korean parent company. You could say Kurzweil is like the Hyundai of the keyboard world.
The K2000 uses V.A.S.T. (Variable Architecture Synthesis Technology) which allows you to take any multi-sample, noise or waveform and process it using just about any synthesis technique. The source of these multi-samples are from the 8MB of ROM which hold tons of authentic and superb quality samples. The internal processing is 32-bit with 18-bit DACs. The K2000 uses 31 sound-shaping algorithms to provide a variety of resonant filters, EQs, continuous panning, amplitude modulation, crossfade, distortion, digital wrap, waveshaper, pulse width modulation, high frequency enhancement, low frequency oscillators, hard sync oscillators and mixing oscillators, all with real-time MIDI control. – www.vintagesynth.com
Ray Kurzweil is also a well known futurist with a pretty interesting track record. This week the NYTimes ran an article about Kurzweil's latest predictions titled, "The Future Is Now? Pretty Soon, at Least". Kurzweil says we will soon have the magic eat whatever you want and don't get fat pill, cheap solar energy and within a decade we will fuse with technology and live forever. He calls that last bit "the Singularity". Check out his book, "The Singularity Is Near: When Humans Transcend Biology": click here.
About 20 years ago he predicted blind people would be able to read anything by using a mobile device. knfbReading Technologies, Inc. in partnership with Kurzweil now sells such a product. Check it out here: www.knfbreader.com
So a name we all associate with a special synth is jumping around in the future. Do you own a Kurzweil workstation keyboard? Do you like them? Did you own one and miss it now?
Kurzweil on ebay: click here
Photo by Michael Lutch. Courtesy of Kurzweil Technologies, Inc.


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James Kraig Kahler allegedly arranged a threesome with his wife and another woman, then snapped after it led to full-fledged affair
JOHN HANNA | Associated Press
LYNDON, Kan. — A former city utilities administrator accused of killing his estranged wife and three other family members in Kansas had encouraged his spouse to have a sexual relationship with a Texas woman but also was an abusive husband, the murdered wife's lover testified Wednesday in his capital murder trial.
Prosecutors called Sunny Reese, of Weatherford, Texas, as a witness against James Kraig Kahler, who's charged in the shootings of his estranged wife, their two teenage daughters and his wife's grandmother. He could face the death penalty if convicted of the slayings, which occurred the weekend after Thanksgiving 2009 in the grandmother's home just outside Burlingame, a town of some 930 residents about 20 miles south of Topeka.
Defense attorneys contend that Kahler snapped mentally because his wife was having an affair with Reese and was pursuing a divorce. Kahler, 48, was utilities director in Weatherford, Texas, before becoming water department director in Columbia, Mo., in 2008, though he lived outside Topeka at the time of the killings.
But Reese testified that Kahler, who often went by his middle name, consented to her sexual relationship with his wife, Karen, before it began in 2008. She testified that he once gave the two women roses at the same time.
"Kraig knew for the get-go and was very accepting," Reese testified. Later, she told jurors, "He agreed to everything. He was very pleased."
Reese also has been subpoenaed as a witness by Kahler's attorneys and could testify again when the defense presents its case. Kahler's attorneys describe him as mentally ill for months before the shootings, suffering from hallucinations and becoming unable to function at work. The killings occurred several months after he lost his job in Columbia, Mo.
Most of the questions Wednesday in Osage County District Court about the two women's relationship came from defense attorney Thomas Haney, who noted that Reese sent Karen Kahler an email in January 2009 about "how to divorce a narcissist." Haney also had Reese read to jurors a text message she sent to Kahler around that time, telling him, "She doesn't love you, Kraig, not like you think she does."
As for Kahler consenting to the lesbian affair, Haney said, "To keep his marriage alive, he was willing to consent to a sexual relationship between her and you."
Haney has repeatedly described the defendant as a loving father and husband. But Reese called the Kahler marriage "a very abusive relationship," saying she'd heard of screaming matches between them. But Reese acknowledged during questioning by Haney that she'd not witnessed incidents herself but based her comments on what Karen Kahler had told her.
"I wanted to protect her," Reese testified. "I thought I could."
The victims of the shootings were: Karen Kahler, 44; her grandmother, Dorothy Wight, 89, and the Kahlers' daughters, Emily, 18, and Lauren, 16. Emily died in the home, and the other three victims were transported to a Topeka hospital before dying.
The Kahlers also had a son, Sean, now 12, who was at the scene of the shootings but fled without being physically injured. He testified Monday, the trial's first day, that he saw his father shoot his mother.
Law enforcement and emergency medical personnel have said that Wight and Lauren Kahler also identified Kraig Kahler as the gunman before they died. Prosecutors have spent much of their time presenting physical evidence tying him to the shootings.
Prosecutors played a recording for jurors Wednesday of an emergency call, triggered by an alarm to a private security and medical emergency monitoring system Wight had in her home. The recording, improved by a law enforcement technician, featured a young woman screaming, "Somebody's going to kill us!" and, in answer to an operator's statement, "He's in the home!"
Reese identified the voice as Lauren Kahler's and bowed her head as she listened to the half-minute recording, shaking at times. Reese appeared nervous throughout her testimony and was often wide-eyed.
But Reese testified that Kahler wanted to bring her into his sexual relations with his wife, proposing it in a text message as the lesbian relationship started.
Karen Kahler's divorce attorney testified Tuesday that she had told him Kahler had proposed such an arrangement. The attorney, Dan Pingelton, of Columbia, said Karen Kahler told him that her husband had brought the two women together, but Reese said they'd known each other since 2006 from working as fitness trainers at a Weatherford, Texas, gym.
Haney noted that no one has produced a copy of a text message from Kahler to Reese, seeking a sexual relationship with the two women.
Haney asked Reese, "You don't have anything other than your testimony today that Kraig Kahler suggested a threesome?"
Reese replied, "No sir. It's my word."


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I finally got around to checking out The Shops at Target in person and boy, was I impressed! The Webster at Target has some seriously cute offerings inspired by the Miami-based boutique. I had the chance to see them at a press preview months ago but it's different seeing them at an actual store, and I was pleasantly surprised by how full the racks were and by the wide selection of items.
The white eyelet dress and colorful navy and coral print shift (shown below) were two of my favorite pieces (you can't tell from the photo but they looked really nice in person). I could see either of these dresses working for a ton of spring/summer events, from graduations to bridal showers.
The breezy cotton tops had lovely embroidered detailing (close-ups after the jump) that rival tops at other stores that cost two or three times as much. I also loved the navy blazer, navy eyelet skirt, maxi dresses and geometric print wrap dress (which was unfortunately polyester - great for traveling since it won't wrinkle but awful for hot weather).
I didn't buy anything on this trip because I was too busy taking photographs, ran out of time and had to jet. But I'll be back to scoop up a thing or two.
Have you seen and/or shopped The Webster at Target? P.S. I was equally impressed with the accessories; I'll share those photos in a separate post.
Shop it online:
Continue reading "Off the Rack: The Webster at ... »


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Nice and comfortable hotel close to the airport
Better than I expected. Nice and comfortable room with a spacious parking lot It is an ideal place to stay don't matter if you are touristing in Miami or in a business trip
A verified traveler from Curitiba stayed at EB Hotel Miami
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Was confortable, clean and very gentleman staff. The location is great.
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The best hotel experience of my life!
The 1 Hotel was the perfect place to stay for our Honeymoon getaway! The staff is beyond fantastic! From the moment we walked in until the moment we left, we were treated like family. Everyone was so nice and accommodating. We had our service dog with us and everyone was incredibly spectacular to her. Our room was gorgeous--we booked the suite with a balcony. It was fully equipped with everything we needed--including a washer/dryer! I've been telling everyone I know about this hotel and how truly dreamy it is.
A verified traveler from brooklyn, new york stayed at 1 Hotel South Beach
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Excellent hotel for Miami airport
Ramada is excellent option for Miami AirPort, its room is confortable and nice, it's pool nice and the breakfast delicious 😋
A verified traveler from orlando stayed at Ramada Miami Springs / Miami International Airport
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Beautiful south beach experience
Very good value. Beautifully situated in the heart of south beach. Right around the corner from a shop where they make handrolled cigars. Tons of eateries. Perfect choice for a weekend getaway
A verified traveler from long island stayed at Dream South Beach
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It was clean and fresh, friendly helpful staff, extremely hot water, very pleased with service.
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Small but elegant rooms
Room was very nice with older-style furniture, very classy. They welcomed you and staff was helpful and friendly. Had a lovely view of the ocean.
A verified traveler stayed at The National Hotel, An Oceanfront Resort
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Great place for a quick business trip to Miami
Had to fly to Miami ahead of Irma to do some business and needed a good hotel near the airport. This was the perfect place. I was able to get in, get my business taken care of and get out quickly. Wonderful staff and great facilities. Wish I could of just stayed and relaxed for 2-3 days. I really could of slept in late in that bed. Thanks for a great experience!
A verified traveler from Guatemala stayed at Best Western Plus Miami Airport North Hotel & Suites
Posted 2 weeks ago
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Infusium (Moisture)ologie Conditioner Reviews
Animal TestingThey test on animals, so I would not recommend this product or anything else by Infusium — 6 years, 10 months agoKatatonia | 191 reviews
This review is: Helpful | Not helpful | Inappropriate | 0 of 3 people said helpful
Infusium 23 classically good!I love this one...and my hairstylist in NYC agrees..makes it smooth and looks less damaged — 4 years, 10 months agoKarine | 15 reviews
Discontinued?I finally found a shampoo and conditioner I will be using for a very long time. I would always always buy different kinds of shampoo, each one claiming to "de-frizz" or "add moisture", etc. Infusium 23 (Moisture)ologie actually did exactly what it claimed to do. This is the first shampoo that I have bought, and finished and ran out to the store to buy again. It smells great, works fantastic, clean...read mores the junk out of my hair and leaves it smooth and healthy. People have been complimenting me on my shiny, healthy looking hair. I am impressed and obsessed!!!! — 6 years, 7 months agoPink52087 | 10 reviews
Worked great for me...I think this product is pretty good. I have very dry damaged hair from my many years of coloring and bleaching. It provided the moisture I wanted and left my hair soft and really easy to style. I have and will continue to recommend this product to friends and family. — 7 years, 7 months agoangelia79 | 1 reviews
This review is: Helpful | Not helpful | Inappropriate | 2 of 2 people said helpful
The BEST product I've found..since 1990I love their shampoo, conditioner and leave-in treatment!!! I've used this production since 1990 and It has never given me a single problem. People comment on how good my hair smells, looks and soft if is. I even use it on all my horses too!! This is the best product by far that would equal any salon product out there...I've got horses with coarse manes...thick tails and after one shampoo, con...read mored'n, spray of the leave in treatment..their show ready. I've even seeen hair growth increase with using this on me and my horses. Thank you and I will always be a loyal customer. I use moisturizing formula in the year, and switch to friz control during the winter, so static is not an issue. K.Wallace Texas — 7 years, 7 months agoKwallace | 1 reviews
This review is: Helpful | Not helpful | Inappropriate | 3 of 3 people said helpful
It's the best!I have tried nearly every brand of shampoo and conditioner known to man. I asked two of the ladies at work how they got their hair so shiny. Their answer was Infusium shampoo and conditioner. I marched right out and purchased both that night. I washed and conditioned my hair, as usual, in the morning and off to work I went. As soon as I walked in the door somebody commented on how shiny and healthy my hair looked. Needless to say I am hooked. — 7 years, 7 months agoKristin1947 | 1 reviews
This review is: Helpful | Not helpful | Inappropriate | 3 of 3 people said helpful
INfusiumAll these products are fantastic for the money — 7 years, 9 months agobornincalif | 73 reviews
This review is: Helpful | Not helpful | Inappropriate | 1 of 2 people said helpful
Soft silky hair at a drugstore priceI've been using Pantene Pro-V for years, but decided to try the Infusium 23 (Moisture)ologie line. This conditioner is so AMAZING that I've permanently switched. It gets my hair silky and soft like never before. I get compliments all the time, especially about the way it smells. I highly recommend it. — 7 years, 9 months agopijan52 | 4 reviews
This review is: Helpful | Not helpful | Inappropriate | 1 of 1 people said helpful
Good daily conditionerI use this conditioner daily, it works well and doesn't leave my hair greasy. — 7 years, 9 months agoann1988 | 14 reviews
Love it!I have very thick, poofy hair. Its to my waist which means everything from my shoulders down is "old" hair. Older hair gets damaged more easily and you need a really good conditioner for it. I hate been looking for YEARS and have tried everything from Paul Mitchell to TIGI. None of those worked as well as this. No more swapping conditioners for me! — 7 years, 10 months agoshanna2104 | 22 reviews
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Tons of new images and details inside...
JAKKS TNA Action Figure Reveal at Slammiversary Celebration
For a great selection of sports action figures and collectibles, check out BigBadToyStore.com, ToyWiz.com, and BriansToys.com.
For a great selection of pop culture t-shirts and apparel, check out TShirtGurus.com and 80sTees.com.
TNA Wrestling Superstars "The Icon" Sting, "The King of the Mountain" Jeff Jarrett, "The Phenomenal" AJ Styles, Olympic Gold Medalist Kurt Angle, "The Samoan Submission Machine" Samoa Joe and Suicide, along with TNA President Dixie Carter, debuted their TNA action figures from JAKKS Pacific, Inc at the Slammiversary Celebration fan event in Orlando, FL, this past weekend.
JAKKS unveiled Wave 1 of its TNA action figures to fans who had a chance to meet, greet and get autographs from each of the wrestlers. TNA Wrestling's Jeremy Borash, Don West and SoCal Val hosted the event.
Slated to launch this summer at Toys"R"Us, Kmart and Target stores nationwide, TNA Wave 1 includes:
TNA Deluxe Impact
The deluxe 7" TNA wrestling figures are fully articulated to perform all signature moves. Featuring TNA talent with Real Scan technology sculpts. Wave 1 includes AJ Styles, Jeff Jarrett, Kurt Angle, Samoa Joe, Sting and Suicide. SRP$ 8.99
TNA Legends of the Ring
The ultimate COLLECTIBLE line of 7" wrestling figures targets collectors with complete authenticity, detailed sculpts & great deco. The deluxe articulated figures replicate authentic wrestling moves, feature Real Scan technology and deco based on iconic outfits & unique appearances. Legends of the Ring celebrates the best wrestling superstars of all time with top talent from TNA, NWA and other top wrestling organizations, including never-before-released superstars. Wave 1 includes Sting (NWA), Kevin Nash, Kurt Angle, Hulk Hogan (NWA), and Jeff Jarrett (NWA). SRP $10.99
TNA Cross the Line Deluxe Figures 2-packs
2 deluxe TNA action figures in 1 pack with deluxe packaging, complete authenticity & detailed sculpts. Fully articulated to replicate real wrestling moves! Wave 1 features Scott Steiner vs Kevin Nash, James Storm vs Robert Roode, Mick Foley vs Samoa Joe. SRP $14.99
TNA Six-Sided Ring
TheTNA Six-Sided Ring playset is an authentic replica of real TNA ring and is the perfect way to stage wrestling events and display your action figures. The hexagon ring has all essential TNA ring elements in the trademark 6-sided ring design.
Source: Edited JAKKS PR


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Pacers trade Mahinmi, Copeland, Watson
Kings trade Marcus Thornton, Travis Outlaw, Jimmer, Isaiah Thomas
Hawks trade John Jenkins (23rd pick in 2012), and Lou Williams
Pacers acquire Thornton, Thomas
Hawks acquire Jimmer, Outlaw
Kings acquire Copeland, Ian, Lou Williams, Jenkins, CJ Watson
Indy adds offensive punch to our bench for the playoff run. Plus, it allows us to POSSIBLY explore trading George Hill in the Summer, and sign Isaiah and Lance. Marcus is off our books next Summer.
Hawks add a more versatile forward with Outlaw. Jimmer would be good for them since he can shoot and handle. And looks for his offense a lot.
Kings get a pass first PG in Lou Williams. Which rumor has it is what they're looking for these days. Jenkins will get a chance to play in Sac's system behind McLemore. Copeland and Landry would be a versatile backup forward combo.


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A SWANMORE College pupil has been accepted into the UK's leading youth brass band.
Rebecca Charters, 15, will attend this year's summer course with the National Youth Brass Band of Great Britain, which gives young instrumentalists the chance to perform advanced music and receive high quality teaching.
It takes place on August 2-9 at Wymondham College in Norfolk and is celebrating its 62nd anniversary.
Cornet player Rebecca, of Waltham Chase, said: "I'm very pleased. I haven't been in the youth band before and the summer course has got great teachers. I'm excited to start."
The teenager also plays with Otterbourne Brass alongside her mother Sonja Charters.
Mrs Charters said: "We as parents are obviously extremely proud of her. She puts in a massive amount of time and practise inbetween all her school work. Becca takes everything in her stride."
The Guest Conductor is Major Michelle Rakers, of the United States Marine Band, and conductor and composer Bramwell Tovey is the artistic director.
The course will finish with two concerts – the first at Beccles Church in Suffolk, and the second at Regent Hall in London.


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Don't miss out on a lower price on this hotel
Get an overview of this hotelFamily-friendly Cairo hotel in Downtown Cairo, near Egyptian Museum
Popular property highlights
This family-friendly Cairo hotel is located on the boardwalk, within 1 mi (2 km) of Tahrir Square, Egyptian Museum, and Cairo Tower. Coptic Museum and Museum of Islamic Art are also within 2 mi (3 km).
Hotel Features
Along with 2 restaurants, this hotel has a full-service spa and an outdoor pool. Free WiFi in public areas and a free manager's reception are also provided. Other amenities include 2 bars/lounges, a fitness center, and a poolside bar.
Room Amenities
All 191 soundproofed rooms provide free WiFi, free wired Internet, and memory foam beds. Guests can enjoy jetted tubs, and other standard amenities include MP3 docks, 24-hour room service, and LCD TVs.
Guest Highlights
- Excellent breakfast
- High-quality beds
- Good location
- Pool
- Helpful staff
- Guestroom size
Kempinski Nile Hotel Cairo
Hotel Amenities
Hotel Amenities
Kempinski Nile Hotel Cairo features a full-service spa, 2 spa tubs, an outdoor pool, and a sauna. Dining is available at one of the hotel's 2 restaurants. Guests can unwind with a drink at one of the hotel's bars, which include 2 bars/lounges and a poolside bar. Guests can enjoy a complimentary manager's reception on select days. Wired and wireless Internet access is complimentary.
Business amenities at this 5-star property consist of a 24-hour business center, limo/town car service, and meeting rooms. Event facilities measuring 1055 square feet (98 square meters) include a conference center. This Art Deco hotel also offers a fitness center, a rooftop terrace, and spa services. For a surcharge, the property provides a roundtrip airport shuttle (available 24 hours), a train station pick-up service, and a train station drop-off service. Limited onsite parking is available on a first-come, first-served basis (surcharge).
Kempinski Nile Hotel Cairo has designated areas for smoking.
- 24-hour front desk
- Babysitting or childcare (surcharge)
- Airport transportation (surcharge)
- Number of floors - 11
- ATM/banking
- Breakfast available (surcharge)
- Valet parking (surcharge)
- Coffee/tea in common areas
- Concierge services
- Dry cleaning/laundry service
- Elevator/lift
- Express check-out
- Fitness facilities
- Full-service spa
- Free WiFi
- Free wired Internet
- Designated smoking areas
- Tours/ticket assistance
- Train station drop-off (surcharge)
- Number of spa tubs - 2
- Total number of rooms - 191
- Laundry facilities
- Limo or Town Car service available
- Spa treatment room(s)
- Meeting rooms 4
- Multilingual staff
- Parking (limited spaces)
- Poolside bar
- Luggage storage
- Safe-deposit box at front desk
- Number of restaurants - 2
- 24-hour business center
- Conference space size (feet) - 1055
- Number of meeting rooms - 3
- Free reception
- Train station pickup (surcharge)
- Sauna
- Number of buildings/towers - 1
- Spa services on site
- Steam room
- Outdoor pool
- Number of bars/lounges - 2
- Free newspapers in lobby
- Conference space
- Pool umbrellas
- Rooftop terrace
- Conference center
- Conference space size (meters) - 98
- Wedding services
Available in all rooms: Free WiFi , Free wired Internet
Available in some public areas: Free WiFi , Free wired Internet
Valet parking (surcharge), Parking (limited spaces)
Room Amenities
- Premium TV channels
- Video-game console
- Hypo-allergenic bedding available
- Air conditioning
- Free newspaper
- Coffee/tea maker
- Free bottled water
- Minibar (stocked, some free items)
- DVD player
- Daily housekeeping
- Turndown service
- Private bathroom
- Designer toiletries
- Bathrobes
- Free toiletries
- Hair dryer
- Jetted bathtub
- Shower/tub combination
- Bidet
- In-room massage available
- Slippers
- Iron/ironing board (on request)
- In-room climate control (air conditioning)
- Soundproofed rooms
- Phone
- Free WiFi
- Free wired Internet
- Dial-up Internet access (surcharge)
- Rainfall showerhead
- Room service (24 hours)
- Down comforter
- Egyptian-cotton sheets
- Memory foam mattress
- Pillow menu
- Free cribs/infant beds
- Rollaway/extra beds (surcharge)
- In-room childcare (surcharge)
- Desk
- In-room safe (laptop compatible)
- Satellite TV service
- Blackout drapes/curtains
- MP3 docking station
- Individually furnished
- Individually decorated
Where to Eat
The hotel offers a complimentary manager's reception on select days.
Osmanly - This theme restaurant specializes in Turkish cuisine and serves lunch and dinner. Open daily.
The Blue - Overlooking the ocean, this restaurant specializes in Italian cuisine and serves breakfast, lunch, and dinner. Open daily.
24-hour room service is available.
Nearby Things to Do
2 spa tubs are on site along with an outdoor pool. Other recreational amenities include a sauna and a fitness center.
Spa Services
Kempinski,the Spa has 4 treatment rooms including rooms for couples. Services include massages, facials, body wraps, and body scrubs. The spa is equipped with a sauna and a spa tub.
- Body treatments
- Facial treatments
- Number of treatment rooms 4
- Couples treatment rooms
- Manicures and pedicures
- Body wraps
- Body scrubs
- Sauna in spa
- Spa tub in spa
- Massages
If you have requests for specific accessibility needs, please note them at check-out when you book your room.
- Accessible bathroom
- In-room accessibility
- Roll-in shower
Hotel Policies
Minimum check-in age is 18
Check-in time ends at 5:30 AM
Check-in time starts at 2 PM
Special check-in instructions:
Check-out time is noon
Payment types
Children and extra beds
- Children are welcome.
- Kids stay free! Children 12 years old and younger stay free when using existing bedding.
- Rollaway/extra beds are available for USD 35.00 per night.
- Free cribs (infant beds)!
- Pets not allowed
You need to know
Extra-person charges may apply and vary depending on property policy.
- Minimum Spring Break check-in age is 18 years old.
Optional extras
- Fee for buffet breakfast: USD 13 per person (approximately)
- Airport shuttle fee: USD 75 per vehicle (one way)
- Rollaway bed fee: USD 35.00 per night
Hotel Name
- Cairo Kempinski
- Cairo Kempinski Hotel
- Cairo Nile Hotel
- Kempinski Hotel Nile
- Kempinski Hotel Nile Cairo
- Kempinski Nile
- Kempinski Nile Cairo
- Kempinski Nile Hotel
- Kempinski Nile Hotel Cairo
- Nile Kempinski
We should mention
This property offers transfers from the airport and train station (surcharges may apply). Guests must contact the property with arrival details before travel, using the contact information on the booking confirmation. Children 12 years old and younger stay free when occupying the parent or guardian's room, using existing bedding. Only registered guests are allowed in the guestrooms.


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Lancaster County Sheriff's Deputies are looking for a pair of concrete lion statues and the person who stole it.
Sheriff Terry Wagner says the first statue went missing sometime between November 20th and the 25th from a house in Prairie Home on North 149th Street.
Then, overnight Sunday, the second lion statue was taken.
Each statue weighs 200 pounds and is two feet tall.
The owner, who did not want to be identified, says the statues have been in front of his house for 23 years.
"They were just a nice piece of decoration. It's kind of a funny feeling because I would have never sold them," the owner said. "It's disappointing. I wouldn't have thought... I thought we were safe out here in the country."
The owner tried to move one of the statues after the first was taken, but it was too heavy.
He believes the first statue was stole while he was on a bike ride and the second when he was working just feet away.
"It makes me ask a lot of questions, and of course it's Christmas time, so you ask, how could somebody steal something at Christmas time," he said.
The owner reported finding a note on his truck about a month before the lions disappeared.
Deputies are looking into that, but are also looking for any leads.
Call Crimestoppers with any information.


text: This news item expired on 9/30/2013, so the information below could be outdated or incorrect. Local Boy & Girl Scouts Encourage Citizens to Tip & Toss! With all the rain we've been getting lately, the mosquito population in Buncombe County seems like it is at an all time high. Buncombe County Health and Human Services, along with some local boy and girl scouts, have a message for you to help combat these pesky creatures.


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Not too far back I upgraded to McAfee Total Protection and everthing was fine. Now whenever a scan is initiated the McAfee window is greyed out. (I can get to HELP and HOME though) and the scan never completes. I recently tried to download an update and rec'd the message that they could find no McAfee products on my system.
I've tried and tried but found no way to contact McAfee in regards to this problem. Any help out there?