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The Project
DNA origami structures are comprised of two basic components. The first, referred to as the scaffold, is M13mp18 bacteriophage E. Coli single stranded DNA (ssDNA), which has a known amino acid sequence, and hundreds of smaller single stranded pieces of DNA (30-50 base pairs in length), referred to as staples. The principles of Watson-Crick base pairing enable the precise design of staples that will bind with specific regions of the scaffold in a piece-wise fashion. The regions of the scaffold that are connected need not be adjacent in the sequence of the scaffold; however, after the staple binding these sequences will be spatially adjacent. In its most basic form, DNA origami is making use of DNA as a rope that knows how to tie itself in knots in order to form rigid shapes.
Triangles are a huge part of the world, as we know it. They can be found in high strength structures in the form of trusses, woven into spider webs, in the patchwork of a soccer ball, and even in the artwork of Matthew W. Moore. The versatility of triangles in nature inspired our group to explore their capabilities on the nano scale. The ultimate goal is to use triangles, combined into parallelograms, as the common factor in the construction of larger, more complex nano structures. Providing this framework allows for streamlining the formation of various triangle based objects as well as the future ability to shift between various objects via the parallelogram intermediate.
Previous DNA origami research has illustrated a wide array of 3D structures. Typically, folding multiple objects requires ordering a new set of DNA components for each desired structure. This project seeks to overcome this limitation by developing a hierarchical assembly framework where multiple 3D shapes can be constructed from a single base DNA origami structure. The basic shape is constructed by folding four equilateral triangles from a single DNA origami scaffold and then arranging them into a parallelogram. Schematics were created to fold these parallelograms into four nanoscale container-like shapes: a tetrahedron, an octahedron, an icosahedron, and a wheel. These final shapes are composed of triangles joined by double stranded DNA connections that can be disrupted utilizing DNA strand displacement to ultimately reconfigure a given shape into a different 3D shape (i.e. reconfigure an octahedron to an icosahedron). This project will enable an economic framework to fabrication of multiple DNA origami structures. Furthermore, this approach could be used to develop DNA structures that can reconfigure in response to a biological stimulus, for example cancer cell microenvironments, for drug delivery applications.
The Methods
The Concept
DNA origami structures are time consuming and costly to build. Each structure requires a new design and thus represents an investment of many hours and as much as one thousand dollars. Our goal was to minimize the cost and time investment of designing a DNA origami structure by creating a single simple structure that has the ability to combine with other simple structures in various ways to make many devices of different sizes and shapes.
We designed a modular structure, a parallelogram composed of four equilateral triangles that can self-assemble into a variety of shapes depending on the addition of connecting staples. These connecting staples, hereafter called external staples, were used to connect different base shapes together to form larger shapes.
Figure 1: one version of the base shape
In figure 1, the numbers 1-12 correspond to potential locations for external staples. Connections can be made between any numbered side, within the same parallelogram or between two parallelograms, to form different shapes. The connections within the parallelogram are also modular. The form shown above is the form 2 parallelogram. The other forms are shown on the next page.
Figure 2: Possible parallelogram conformations, only form 2 and form 3 are used in the formation of other shapes
The parallelograms 2 and 3 are inverses of each other, meaning that the sides that are masked (used to make internal connections within the parallelogram shape) in form 2 are presented to solution in form 3 and vice versa. Additionally, the form 1 parallelogram was never physically manufactured. It merely serves as a useful visual intermediate in the folding of form 3.
Our design differs from traditional DNA origami in the use of "external staples." Traditionally, staples are complementary to specific sequences of the scaffold. The scaffold-staple binding brings sequences of the scaffold together spatially and holds them there. In contrast, the external staples in our design never bind to the scaffold but instead bring together specific staples sequences that are external to other structures. These external staples are 18 base pairs in length. They are complementary to single-stranded DNA that hangs off the edge of sides of the equilateral triangles. These staples, hereafter called polymerization staples, are 10 base pairs in length and allow modularity in the design. Essentially, the hangover staples are unique sequences that provide attachment points between triangles or parallelograms. Our design incorporates 96 unique polymerization staples and over 400 external staples. External staples are unique in that they are complementary to specific polymerization staples. Each external staples is complementary to 9 base pairs from one polymerization staples and 9 base pairs from another polymerization staples (the polymerization staples are 10 base pairs each to allow some slack in the connection sites). When the external staples are put in solution they bind to the polymerization staples and draw the sides attached to the polymerization staples together in order to form the desired shape.
Each side of an equilateral triangle contains 8 polymerization staples. These polymerization staples are named uniquely. Each side is numbered 1-12. Each half of a trapezoid is labeled a-f. The outermost four connection points on each side are labeled "out" while the four inner connections are labeled "in." Polymerization staples are paired and extend from the structure at adjacent loci. The staple that presents the 5' end is therefore designated as 5' while the polymerization staples that presents the 3' end is designated 3'. This naming convention can be seen in the diagram below.
Figure 3: Naming convention for external and polymerization staples (4a out 5' and 4b in 3' are examples of how the naming convention works)
Figure 4: Layout of the design as seen in caDNAno in 2 dimensions. The 8 connections and labeling displayed in figure 3 are present in each of the 12 sides displayed above.
Figure 4 depicts the layout of the design as it is seen in caDNAno. The red lines represent scaffold routing at the pivot points that allow the formation of the 3 forms of the parallelograms that can be seen in figure 2. Each column of 3 "triangles" (note that the isosceles triangles in figure 4 above more closely resemble trapezoids) folds to form an equilateral triangle. A close up of the equilateral triangle scaffold routing can be seen below in figure 5.
Figure 5: The 5 rows mentioned above can be seen here. The nonlinear segments correspond to single stranded scaffold that joins the 5 rows. Note that each row of the trapezoid is separated only by the diameter of a DNA helix.
Detailed design description
The base shape consists of four equilateral triangles made out of a single scaffold 8064 base pairs in length. Each equilateral triangle consists of three identical trapezoids that are joined by ssDNA at the center and has a side length of 52.77 nm. The trapezoids are in turn composed of five parallel "rows" of DNA double helices. Each row is 24 base pairs shorter than the row above it: for example the top row contains 182 base pairs with 158 contributing to the structure (182 – 12*2) for a total length of 52.77 nm. The 24 base pair reduction between each row yields the 30ᵒ angle that is necessary to form the equilateral triangles when the trapezoids are joined together. This 30ᵒ angle and 12 base pair reduction between each row of the trapezoid necessitated 12 base pairs of ssDNA in order to form the junction without causing stress in the design.
Each equilateral triangle is attached to one or two other through single stranded scaffold. These connections are loose (8 singles stranded base pairs is approximately 5.6 nm) and would allow the triangles to move freely without the addition of more structural staples. These additional connections transform the loosely connected equilateral triangles into the parallelogram. These structural staples were provided in two forms. One form will be called internal bridge connections. These staples were "hardwired", meaning that these connections could only be formed in one way. In other words these staples follow the traditional DNA origami method of complementarity with specific segments of DNA thereby drawing those segments together. The other form, designated as the external bridge design, relied on external staples to form the connections that would form the base shape.
Prestocks and Working Stocks
Working stocks and prestocks were used to simplify the folding processes. A prestock is a set of staples that have been combined because they are all used to create the same module of the design . For example, all staples used to create the internal connections of the parallelogram were added to the same prestock because they all serve the purpose of creating internal connections in the sub-triangle base shapes. This technique saved time and effort when working in the lab. When a structure is to be made and all of the internal connections needed to be added to a working stock, one needs to simply take from a single prestock instead of dozens of single oligos.
Working stocks are combinations of specific prestocks and additional staples in a single tube to bring together all staples required for a desired folding reaction. An example of a working stock would be the combination of the internal staples in the previously mentioned prestock with other prestocks of the equilateral triangles to create the base parallelogram shape. When preparing the sample for the folding reaction, a certain volume of working stock was used to produce the shape, while the remainder of the working stock was stored at 4° C to allow for future folding reactions.
Formation of parallelogram form 2 utilizing the internal bridge.
Formation of parallelogram form 2 utilizing the external bridge.
Formation of parallelogram form 3 utilizing the internal bridge. It was observed that this bridge method resulted in a lower yield.
Formation of parallelogram form 3 utilizing the external bridge. It was observed that this bridge method resulted in the lowest yield of the four parallelogram formations.
Step 1 in the formation of the octahedron with the use of parallelogram form 2 and the internal bridge. This working stock also contains the 10-12 external connection staples.
Step 1 in the formation of the octahedron with the use of parallelogram form 2 and the external bridge. This working stock also contains the 10-12 external connection staples.
Step 1 in the formation of the octahedron with the use of parallelogram form 3 and the internal bridge along with the addition of the 10-12 external connection staples.
Step 1 in the formation of the octahedron with the use of parallelogram form 3 and the external bridge along with the addition of the 10-12 external connection staples.
Formation of the tetrahedron with the use of parallelogram form 2 and the internal bridge along with the addition of all external connections staples.
Formation of the tetrahedron with the use of parallelogram form 2 and the external bridge along with the addition of all external connection staples.
Formation of the wheel with the use of parallelogram form 2 and the internal bridge along with the addition of all external connection staples. It was discovered that this concerted process of formation was not successful; therefore the formation of the wheel was separated into three steps. This is described in detail in shape formation.
Formation of the wheel with the use of parallelogram form 2 and the external bridge along with the addition of all external staples. It was discovered that this concerted process of formation was not successful; therefore the formation of the wheel was separated into three steps. This is described in detail in shape formation.
Polymerization and External Staples
The 96 polymerization staples need to be unique sequences. Additionally, the design incorporates 60 single-stranded scaffold components to form the connections between the rows within a trapezoid. In order to avoid difficulties with unwanted binding between polymerization staples or external staples, the 96 polymerization staples needed to each be different from the 60 single-stranded scaffold sequences and from the reverse complement of each of these 60 sequences. Moreover, each polymerization staple adds additional restrictions. The problem is further complicated by the fact that multiple complementary base pairs will weakly bind to each other locally even if the entire strand is not complementary. We therefore limited our strands to have less than 5 consecutive complementary base pairs out of 10 base pair external staples sequences. 4n different sequences can be created for a sequence with n base pairs. In our case where the external staples contain 10 base pairs, 1,048,576 sequences are possible. However, out of more than a million options fewer than 400 met the specifications for our external staples. These staples were produced using the program displayed below, written by Andrew Krieger. This program first generated all possible sequences starting with 10 consecutive A's and then created sequences after comparing them to a list of the 60 single-stranded scaffold sequences. Each staple created was stored and then added to the list of disallowed sequences. It is worth noting that the sequences produced differ when the initial input is changed.
Additionally, the relationship between the number of acceptable staples and the parameters (staple length and number of consecutive complementary base pairs allowed) for a given set of parameters is complex. For 4 consecutive complementary staples allowed for strands of 6 base pairs 46 external staples were generated by the program. However, for 4 consecutive complementary staples allowed for strands of 7 base pairs only 36 external staples were generated. We suspect that this decrease in number of staples despite the increase in the strand size is due to the fact that adding a staple adds a four-fold increase the number of possible staples but also adds many more restrictions by increasing the chances that a staple is complementary with one of the disallowed sequences.
Figure 3: Part 1 of the Program that generated the external staples, written by Andrew Krieger
Figure 3: Part 2 of the Program that generated the external staples, written by Andrew Krieger
Shape Formation
Working stocks 9 and 10 (described in Working Stocks and Prestocks LINK) form the tetrahedron with the use of parallelogram form 2 in two different versions; one utilizing the internal bridge and the other the external bridge. The formation occurred in one concerted thermal ramp cycle followed by an agarose gel electrophoreses purification. A schematic for the tetrahedron is shown below.
Step 1: Formation of both forms of the parallelogram with 10-12 external connection staples, which will allow the complementary form to bind. This step creates working stocks 5 through 8, varying in parallelogram form and bridge form. These working stocks were purified through agarose gel electrophoresis prior step 2.
Step 1 – formation of parallelograms: Parallelogram form 2 with internal bridge (working stock 1) as well as parallelogram form 3 with external bridge (working stock 3) were folded. These folded parallelograms then underwent gel electrophoreses.
Step 2 – connection of the top two parallelograms: A dilution of 8-12 external staples was formed and combined with working stocks 1 and 3. This was then incubated over night at 37°C and 400rpm.
Step 3 – connection of the bottom two parallelograms: A dilution of 2-6 external staples was formed and combined with working stocks 1 and 3. The combined parallelograms and staples were then gel purified.
Step 4 – addition of the middle parallelogram to the top two parallelograms: A dilution of 6-6 external staples was formed and combined with icosahedron step 2. This was then again gel purified.
Step 5 – combination of top three parallelograms to the bottom two parallelograms: A dilution was formed of 12-12 external staples and combined with the purified icosahedron steps 3 and 4. This was then incubated over night at 37°C and 400rpm.
Step 6 – linkage of side-to-side connections: A dilution was formed of the external staples for the side-to-side connections (1-5, 3-7, 1-9, 9-10, 7-11) and combined with icosahedron step 5. This was then incubated over night at 37°C and 400rpm. Icosahedron step 6 formed the icosahedron with a seam where the top and bottom had not yet been combined.
Step 7 – linkage of top-to-bottom connections: A dilution of the external staples for top-to-bottom connections (4-5, 2-8, 10-11) was formed and combined with icosahedron step 6. This combination was incubated over night at 37°C and 400rpm. This final step completes the linkages necessary to form the shape of the icosahedron. Because of the small concentration of icosahedrons in this step, gel purification was not performed prior to preparing the carbon coated transmission electron microscope grid for imaging.
Version one was simply a concerted process (working stocks 11 and 12) in which form 2 internal bridge or external bridge was combined with all the external staples. This version was found to not be successful in forming the final shape of the wheel.
Version two and three were accomplished through the following steps. The difference between the two versions was the incubation period between step 2 and step 3 – 30 minutes for version 2 and overnight for version 3.
Step one: Parallelogram form 2 with internal bridge (working stock 1) was folded in a thermal ramp and gel purified.
Step two: A dilution of 7-9 external staples was formed and combined with wheel step one. This combination was then incubated at 37°C and 400rpm for either 30 minutes or overnight depending on the version.
Step three: A dilution of 6-12 and 1-3 external staples was formed and combined with wheel step two and incubated overnight. After incubation, the final wheel sample was again gel purified.
Thermal Ramp
Gel Purification via Agarose Gel Electrophoresis
Once the gel is prepared and the sample is loaded, the gel box is inserted into an ice water bath and a 70V current is applied. Strands of shorter length or structures that are more compact will migrate with greater ease through the agarose gel. This will allow for the 10-fold excess of staples that was added in the design to be separated from the properly folded structures, as well as allow separation between well folded structures and possible dimers and trimers.
Grid Preparation
Thermal Ramp
Design Complications
The previously mentioned external staples were composed of the same sequences in every parallelogram. Although this design allows for self-assembly with the addition of polymerization staples into solution, it also presents difficulty in creating more complex shapes. Suppose we wish to join two form 2 parallelograms. We could make a set of 8 external staples to form a connection between the 6-side and the 12-side of different parallelograms. However, we now have an issue. The parallelogram that bound with its 6-side presents a 12-side to the solution while the parallelogram that bound with its 12-side likewise presents its 6-side to solution. The external staples in solution will thus continue the reaction and cause additional parallelograms to bind to the open sites. These parallelograms also have open binding sites allowing for even more polymerization. This polymerization is uncontrollable and will yield a distribution of unwanted products containing more than the desired two parallelograms.
To avoid this polymerization issue, we instead bind a side to its own equivalent on another connection. In other words if we take the form 2 parallelograms from the previous connection and make a 6-6 connection instead of a 6-12 connection then the parallelograms can only bind once. Once the 6-6 connection has been made between the two parallelograms these bound parallelograms do not present 6-sides to solution, their 6-sides are already bound. This strategy allows control of the folding reaction and will only create two-parallelogram structures.
An additional design problem was encountered due to DNA strand displacement. DNA strand displacement is a technique whereby a strand of DNA (designated A) that is bound to another strand (designated B) can be displaced by a strand of DNA with greater affinity for strand B (designated A'). When strands A and B are in solution they will bind together; however, when strand A' is introduced, it will displace strand A overtime due to random thermal fluctuations and its greater binding affinity for strand B. Although cytosine (C) and guanine (G) have a greater binding energy than adenine (A) and thymine (T), the binding affinity of strand A and A' to B in DNA origami depends primarily on their lengths because their sequences are identical excluding the extended length that A' has over A.
Our design incorporates many single-stranded components to give it the flexibility necessary to form more complex structures. Although our design process ensured that the polymerization staples and single stranded scaffold between the trapezoids were not complementary for more than 5 consecutive base pairs, some binding between the scaffold and polymerization or external staples would occur during the folding process. This binding, however, was transient because the unwanted bindings were eliminated by DNA strand displacement from staples that had higher affinity.
Unfortunately, DNA strand displacement did not work for our external staples. The external staples were designed to bind to 9 single-stranded base pairs on polymerization staples on two faces of the base shape. Thus the fully bound external staple would be bound to 18 base pairs from 2 different polymerization staples. External staples are added in tenfold excess to ensure that all the sites on the structure requiring external staples are used. In this particular case, the excess of staples means that every polymerization staple was bound to an external staple; however, it is far more likely that every polymerization staple would be bound to a separate external staple rather than binding an external staple attached to another parallelogram. Thus every binding site would be full and unable to accept a bond to another parallelogram.
Moving Forward
Our design represents a method for bottom-up assembly of complex structures from DNA origami. It has the potential to save money and time during the design and manufacturing process of DNA origami structure. Moreover, the base shape could be modified to fit other applications by inducing curves in the equilateral triangles or by changing the base shape from a design based on triangles to a design based on squares. This design, and the methodology behind it's construction, has the potential to revolutionize the formation of complex 3-D structures in DNA origami.
The Concept
Detailed Design Description
Prestocks and Working Stocks
Polymerization and External Staples
Shape Formation
Grid Preparation
Cadnano Design
Design Complications
Moving Forward
The Results/Discussion
The project design called for the creation of a base shape parallelogram, tetrahedron, octahedron, icosahedron, and a 3-dimensional wheel. Initially, the base shape parallelograms were imaged. There was a very high yield of the equilateral triangles proving the first step of our design was a success.
The formation of the base shape parallelogram was critical; it was the base unit used to create all other shapes. Since the design called for the parallelogram to contain pivot points, it was hard to view a perfect image of the parallelogram completely flattened out. However, there were several images that appeared to have only three equilateral triangles, with the fourth possibly folded behind its neighboring triangle due to the single stranded slack between them. Even though it was difficult to obtain images of 4 equilateral triangles coming together properly to form the base shape parallelogram, we have been able to show its success because of the high yield of the 3D tetrahedron shape. The tetrahedron was designed to contain exactly one parallelogram folded at the pivot points and imaging of the shape showed a very high yield.
A transmission electron microscope was used to show that the folding reaction was successful in creating the parallelogram base shape. The triangles during polymerization were successfully able to bind to neighboring triangles in the correct order to create parallelograms.
Figure 1: Triangles folded into parallelograms using a MgCl solution during the polymerization process. The TEM picture was taken at 100 nm at 80kV with a magnification of 98000x.
Figure 2: Triangles folded into parallelograms using a MgCl solution during the polymerization process. The TEM picture was taken at 100 nm at 80kV with a magnification of 120000x.
Figure 3: Triangles folded into parallelograms using a MgCl solution during the polymerization process. The TEM picture was taken at 100 nm at 80kV with a magnification of 120000x.
It was noticed, however, that the yield of fully bound parallelograms was very low. Triangles binding in an ordered manner of three or less were a common occurrence. Another common observation was two triangles folding on top of another two triangles. Although four triangles were successfully bound together they did not fall to the surface of the TEM grid in the proper orientation. It was hypothesized that the slack between equilateral triangles allowed them to fold on top of one another, resulting in images that appeared to have only one, two, or three triangles.
It was observed that the parallelogram working stocks were properly synthesized and successfully folded into tetrahedrons. While observing the tetrahedrons it was apparent they had a well-defined three-dimensional shape. Pivot points that were created in the design process were successful due to the nature of the folding. Corners of the tetrahedrons were properly held together with staples and scaffold. From a visual representation it appears that tetrahedrons gave the highest yield of fully polymerized geometric shapes.
Figure 4: Parallelograms folded into tetrahedrons using a MgCl solution during the polymerization process. The TEM picture was taken at 100 nm at 80kV with a magnification of 120000x.
Figure 5: Parallelograms folded into tetrahedrons using a MgCl solution during the polymerization process. The TEM picture was taken at 100 nm at 80kV with a magnification of 68000x.
Figure 6: Parallelograms folded into tetrahedrons using a MgCl solution during the polymerization process. The TEM picture was taken at 100 nm at 80kV with a magnification of 120000x.
After observing the tetrahedrons successfully folding, an experiment was developed to see if the folded devices could be successfully dismantled, and then refolded back to their proper shape. The working stock that contained the folded tetrahedrons was heated overnight at two different temperatures, 35° and 40° C. After being heat-treated the DNA solutions were immediately pipetted onto a TEM grid to prevent the tetrahedrons from refolding before observations of the unfolding could be made.
This experiment showed that the tetrahedrons were successfully able to unfold after being heat treated at 35℃ and 40℃. Although both temperatures were successful in unfolding tetrahedrons into parallelograms, it is apparent that reheating the DNA structures to 40℃ yields greater results of unfolding. We hypothesized that the structures heated to 35° C were able to begin the refolding process while they were being prepared on the TEM grid. By increasing the temperature to 40℃, the structures were at an elevated temperature for a longer period of time, thus reducing their ability to refold while preparing them on the TEM grid.
Figure 7: Tetrahedrons were unfolded by increasing the temperature to 35℃ during the polymerization process. The TEM picture was taken at 100 nm at 80kV with a magnification of 98000x.
Additionally, some of the unfolded tetrahedrons were left over night at 4° C. After they were given ample time to refold, new TEM grids were prepped to show that the structures successful reformed into their tetrahedron shape. The ability to have DNA structures unfold and refold demonstrates how our design can be effectively reused. However, it was observed that working stocks that were heat treated to refold contained a lower yield of geometrically viable shapes. Although perfectly folded tetrahedrons were present in TEM pictures after refolding there was an increase in deformed tetrahedrons. Images showed that the tetrahedrons in the working stock that was heat treated to 40℃ contained a higher yield of properly folded tetrahedrons compared to the heat-treated 35℃ working stock.
Figure 8: Tetrahedrons that were unfolded at a temperature of 35℃ were refolded during the polymerization process. The TEM picture was taken at 100 nm at 80kV with a magnification of 98000x.
Figure 9: Tetrahedrons that were unfolded at a temperature of 40℃ were refolded during the polymerization process. The TEM picture was taken at 100 nm at 80kV with a magnification of 120000x.
Purified versus Non. Purified
An experiment was done to determine if unpurified gels had a greater yield than purified gels. The thought behind this hypothesis is that unpurified gels will have a higher yield because they are essentially unfiltered. By having an unfiltered gel the TEM grid could potentially have a higher yield of synthesized origami shapes. The result concluded that purified gels were more applicable for observing origami shapes. Unpurified gels contained a large amount of impurities, monomers, and deformed origami shapes. The unpurified gel TEM grids were extremely cluttered and were observed to have greater dye absorption. Unpurified gels were observed to have no increase in origami figures; however there was an increase in monomers binding to origami figures yielding an increase in deformed structures. In conclusion purified gels should be the most appropriate choice when viewing DNA origami with a transmission electron microscope.
Once agarose gel electrophoresis is complete utilizing ethidium bromide in staining, the DNA in the gel is viewed on a FOTO/Convertible Dual Wavelength UV light. An example of what would be seen at this point can be viewed in figure 1. This is then imaged through an ethidium bromide filter. As an example, an image of a gel generated from the 60-hour ramp of variations in the folding of the parallelogram can be seen in figure 2. 6 µl of 1kb ladder was loaded into well 1 to be used as a reference to the speed at which our structures travelled in comparison to a control known length segment of DNA and can be seen at the bottom of figure 4. Wells 3 through 10 were loaded with variations of the parallelograms including alternating the concentration of magnesium chloride utilized prior to the folding reaction. The specifics of each well for this image are indicated in figure 3.
In agarose gel electrophoresis, molecules of smaller size migrate with less resistance through the pores of the gel. In creating each shape, staples were added in 10-fold excess, which results in an extremely bright band of surplus staples. The bright band that travelled farther within the gel (the "fast" band) is indicative of properly folded structures while the minor band closer to the well shows the presence of dimer structure. Both of these bands were extracted from the gel for analysis. In order to avoid contaminating the samples, a new sterile scalpel was used to cut out each band from the gel.
Figure 1
Figure 2
Figure 3
Figure 4
The Resources
Ke et. al., Scaffolded DNA Origami of a DNA Tetrahedron Molecular Container. NanoLett. 2009; 9(6): 2445-2447. http://pubs.acs.org/doi/pdf/10.1021/nl901165f
Rothemund et. al., Algorithmic Self-Assembly of DNA Sierpinski Triangles. PLoS Biol. 2004; 2(12): E424. http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.0020424
Rothemund, Paul W. K. Folding DNA to create nanoscale shapes and patterns. Supplementary Notes 1-11. Nature 2006; 440: 297-302. http://www.nature.com/nature/journal/v440/n7082/extref/nature04586-s1.pdf
Rothemund, Paul W. K. Folding DNA to create nanoscale shapes and patterns. Supplementary Note 12. Nature 2006; 440: 297-302. http://www.nature.com/nature/journal/v440/n7082/extref/nature04586-s2.pdf
Rothemund, Paul W. K. Folding DNA to create nanoscale shapes and patterns. Nature 2006; 440: 297-302. http://www.nature.com/nature/journal/v440/n7082/full/nature04586.html
Wei et. al., Complex shapes self-assembled from single-stranded DNA tiles. Nature 2012; 485: 623-626. http://www.nature.com/nature/journal/v485/n7400/full/nature11075.html
Yang et. al., DNA origami with double-stranded DNA as a unified scaffold. ACS Nano. 2012; 6(9): 8209-8215. http://www.ncbi.nlm.nih.gov/pubmed/22830653
Zhao et. al., Organizing DNA origami tiles into larger structures using preformed scaffold frames. Nano Lett. 2011; 11(7): 2997-3002. http://www.ncbi.nlm.nih.gov/pubmed/21682348


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Mostly you hear about horror stories of people and their brushes with technical support and product repair.
Here I want to mention the fantastic service I received from the MP3 player people Rio.
My Rio Karma that I purchased last June suddenly locked up, I could not switch it off and no keypress was recognised. Luckily I had a pin and reset it, this turned it off. When I turned it on again it immediately locked up again.
Next I read the manual and tried doing what this said but again it locked up.
Now Friday Afternoon I sent an email to their technical support and on Saturday morning I received a reply, tried that, no help, emailed again and got another reply, no good, emailed again and this did not help either. He apologised and said I needed an RMA number. Please ring an (0207) number in office hours.
On the Monday I rang, the phone in London was transferred to the Netherlands and after a few questions was given the RMA number together with the address to send the unit to. I was told it would take up to two weeks.
On the Thursday of the following week a courier delivered a replacement Rio Karma. Yes it was within the warranty period my word was taken on this and I had to pay postage to the Netherlands £1.48, but this was how warranty claims should work.
Well done Rio, up till then I had never regretted the purchase and after such good service I still do not. I know it was a different unit returned as mine had a small scratch and this one was perfect.
Anyone else get good service, or is Petta just lucky?
Had a good experience with Dell a few years back.
I bought one of there mini-tower systems, an XPR-400 which to those that know show how long ago this was!
The system arrived as expected and everything worked fine for a couple of days and then the 17" monitor just died. No warning - it just shut down and refused to do anything.
I got onto Dell, who were very helpful and immediately sent out a replacement monitor. As it happened they provided a replacement with a better spec than the original. I'd gone for the basic 17" product - and they replaced it with an excellent Triniton tube monitor.
The computer itself is still gong strong almost 6 years later. Now equiped with 3 hard-drives, the latest a 160Gbyte devive, it forms a cental store for all our music and photos.
My more recent experience with Dell products hasn't been so good....... but you did ask for good stories!
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This product is no longer available for purchase. But you might be interested in the Orion SteadyPix Universal Smartphone Telescope Photo Mount. You can view this product by clicking on the product image or the button below. Thank you.
–The Orion Team
Take beautiful astrophotos through any telescope with your iPhone! With the Orion SteadyPix Telescope Photo Adapter for iPhone, anyone with a telescope and an iPhone can easily take stunning pictures of the night sky.
The SteadyPix iPhone Photo Adapter securely couples an iPhone 4S, 4, 3GS, 3G, or original iPhone to a 1.25" telescope eyepiece so you can take high-magnification pictures through the telescope using the iPhone's built-in camera. The sturdy, steel adapter bracket allows the iPhone's camera lens to be positioned directly over the 1.25" telescope eyepiece eye lens for afocal eyepiece-projection photography. Since the iPhone camera is positioned right where your eye would be, it's easy for anyone to start taking pictures of night sky wonders, no matter how experienced or inexperienced you are in the hobby of astrophotography.
At night, capture high-resolution images of the Moon's cratered surface and take astrophotographic snapshots of bright and interesting planets like ringed Saturn and gigantic Jupiter. With practice and a bit of patience, you can even capture images of some bright deep-space objects and the International Space Station (really)!
The fun doesn't stop at sunrise either. Use the SteadyPix iPhone adapter during daylight hours with a refractor or Cassegrain telescope to take superb high-magnification snapshots of distant wildlife and scenery. With a properly sized solar filter attached to any telescope, you can take daytime snapshots of broiling sunspots on our nearest star with your iPhone.
Another great feature of the Orion SteadyPix Telescope Photo Adapter is how it lets you display a live view of the image projected by your telescope on your iPhone screen. Now you can share beautiful real-time views of the night sky with friends and family by using your iPhone as a mini telescope display monitor! No more lining up to wait for your chance to view, everyone can observe at the same time when using an attached iPhone as a cool mini display screen. The SteadyPix iPhone Photo Adapter can also attach your iPhone to a photo tripod for steady wide-field pictures without a telescope.
The Orion SteadyPix Telescope Photo Adapter for iPhone includes a felt-lined steel eyepiece clamp that fits standard 1.25" telescope eyepieces with housing diameters up to 1.5". A larger clamp for bigger eyepiece housings is sold separately (see suggested accessories).
For best results, you may need to remove your iPhone's protective case before installing in the SteadyPix Telescope Photo Adapter.
Not compatible with iPhone 5. For iPhone 5 users, we recommend our popular Orion SteadyPix Universal Smartphone Telescope Photo Adapter, which is compatible with the iPhone 5.
Apple, the Apple logo, iPhone, iPod, iPad, iPod touch, and iTunes are trademarks of Apple, Inc., registered in the U.S. and other countries.
Limited Warranty against defects in materials or workmanship for one year from date of purchase. This warranty is for the benefit of the original retail purchaser only. For complete warranty details contact us at 800-676-1343.
Orders received by 1pm Eastern Standard Time for in-stock item the same business day. Order received after noon will ship the next business day. When an item is not in-stock we will ship it as soon as it becomes available. Typically in-stock items will ship first and backordered items will follow as soon as they are available. You have the option in check out to request that your order ship complete, if you'd prefer.
This product is available to ship Standard delivery within the 50 US states, APO/DPO/FPO addresses and US territories/protectorates. Delivery is not available to Canada.
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Online or in your mailbox. Check out our colorful catalog, filled with hundreds of quality products.
Please send me email about sales, new products, and astronomy.
Since 1975 Orion Telescopes & Binoculars has been offering telescopes for sale direct to customers, with an unswerving commitment to best quality products, value and unmatched customer care. Our 100% satisfaction guarantee says it all.
Orion offers telescopes for every level: Beginner, Intermediate, Advanced, and Expert. From our entry level beginner telescopes for amateur astronomers to our Dobsonian telescopes to our most advanced Cassegrain telescopes and accessories, you can find the best telescope for you. Because we sell direct, we can offer you tremendous value at a great price. Not sure how to choose a telescope? Orion's Telescope Buyer's Guide is a great place to start.
Orion binoculars are known for quality optics at a great price. We offer binoculars for every viewing interest, including astronomical binoculars, compact binoculars, waterproof binoculars, birding binoculars, and sport and hunting binoculars.
Orion's telescope and astrophotography accessories will enhance your telescope enjoyment without breaking the bank. Expand your viewing experience with accessories ranging from moon filters to power-boosting Barlow lenses to advanced computerized telescope mounts. Capture breathtaking photos with our affordable astrophotography cameras. And when you're stargazing, Orion's telescope cases and covers, observing gear, red LED flashlights, astronomy books and star charts will make your observing sessions more convenient, comfortable and meaningful.
At Orion, we are committed to sharing our knowledge and passion for astronomy and astrophotography with the amateur astronomy community. Visit the Orion Community Center for in-depth information on telescopes, binoculars, and astrophotography. You can find astrophotography "how to" tips and share your best astronomy pictures here. Submit astronomy articles, events, & reviews, and even become a featured Orion customer!


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Pedalling in the footsteps of Joseph Alois Ratzinger – Pope Benedict XVI on the 200km-plus circular Benedict Trail between the river Inn and the river Salzach in south-east Upper Bavaria.
Altötting has been the religious heart of Bavaria for over 1,250 years and, for more than 500 years, has been the foremost centre of pilgrimage to the Virgin Mary in the German-speaking countries. The lime tree planted here by Pope John Paul II in 1980 marks the start and finish of the Benedict Trail. This pilgrimage route links many sites significant to Bavarian popular piety as well as places where the German pope spent his youth. They include Marktl, where Joseph Ratzinger was born, Tittmoning, where he went to kindergarten, and Aschau and Traunstein, where he attended school and the priests' seminary. The trail is also named after St. Benedict, who founded the Benedictine order, and Bavaria as the terra benedicta. Some feel closer to heaven here in this 'blessed land' than elsewhere. People from every corner of society have been raised to the glory of the altar here, whether an aristocrat such as the king's daughter Irmingard, who founded the convent on Frauenchiemsee island, or the son of a peasant, like Brother Konrad from Parzham, who was the gatekeeper at Altötting's Capuchin monastery. The Benedictine motto ora et labora (pray and work) has left its mark on this diverse landscape dotted with abbeys and pilgrimage sites between the Salzach and Inn rivers and the Chiemgau region. Amid this beautiful panoramic countryside, the Benedictines, Cistercians, Franciscans and Augustinians are all represented along the Benedict Trail in the form of proud abbeys, baroque pilgrimage churches and other splendid sacred buildings.
Kaksi hyödyllistä näppäinyhdistelmää selaimen tekstikoon muuttamiseksi:
Suurenna: +
Pienennä: +
Lisäohjeita saat selaimen valmistajalta napsauttamalla kuvaketta:


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By: Matt Moore CBS Sports
As the NBA catches its breath after a hurried rush to the season and a whirlwind All-Star Weekend, we turn our attention to the NBA's stretch run, the second half. We're at the point with the trade deadline looming two weeks away where teams will begin to make moves to either ramp up for a playoffs run or bottom out in rebuilding projects. Will the Heat keep up their pace? Can the Bulls finally get healthy and if so, how good can they be then? Are the Thunder legitimate title contenders? Are the Spurs "back" once again?
So many questions, so little time.
A huge element in the second half of the season is going to be minutes. The best teams will rest their players while desperate teams will have to play to the bone. The Bulls are somehow a great team and a desperate team. They want every advantage including homecourt advantage, and below we'll tell you how that could impact the MVP race.
So we present the Baseline Awards second-half predictions, focused on who will be taking the various trophies for the second half of the season.
Projected Eastern Conference Player of the Second Half: Derrick Rose
LeBron James is the runaway MVP leader, the best player on the planet, and was nearly flawless in the first half, despite the end to that All-Star Game. So why isn't he here? Because the Heat will have a top-two seed locked up by early April, and then you're going to see James take more and more games off as the Heat rest him for the playoffs. What he does between now and then will determine is MVP, but in the meantime, Tom Thibodeau plays his players 35 minutes a game even when they're hurt. Assuming that the All-Star rest helped Rose recover from the back and leg injuries that hampered him this season, he'll get the minutes, he'll get the production, he'll get the win. I expect big things from the reigning MVP in the second half.
Projected Western Conference Player of the Second Half: Kevin Durant
It's terrifying that a kid, a 23-year-old kid, can lead the league in scoring two years in a row and then make the leap. But that's what Durant has done. Everything is better. His efficiency. His productivity. His team. His leadership. His clutch play. And his defense. It's a phenomenal streak the kid's on and there's zero reason to think he'll slow down in the second half. The West is just competitive enough for the Thunder to keep Durant playing nightly and yet not good enough to challenge the Thunder for the top spot in the West (though San Antonio's making a good show of it).
Most-Likely to Succeed: Miami Heat
They have it together. They have no discernible weaknesses, outside of "Can LeBron James succeed in the clutch?" and that's not a regular season concern. The offense is clicking, their chemistry is good, they've had fewer distractions, they're playing at an elite level on both sides of the ball and their injuries haven't been significant. It may be sickening, but it doesn't change the fact that this is the baddest team in the land until further notice.
Least-Likely to Succeed: Charlotte Bobcats
Here's the worst thing about the Bobcats. They don't even have anything of value to firesell. Tyrus Thomas is having a terrible season, Corey Maggette has been injured, D.J. Augustin is just good enough to be their best player and not good enough to draw huge offers on the market. Gerald Henderson may be their best asset and he's too good to move. If they keep this team, they'll be horrible. If they sell off the parts for future components, they'll be terrible. If the lottery doesn't help this team, it's going to be classified as negligible cruelty.
Best Dressed: Indiana Pacers throwbacks
These jerseys are awesome. (Getty Images)
Those blues and yellows are just sharp as all get out.
Worst Dressed: Memphis Grizzlies throwbacks
This, not so much a crime against fashion as a crime against humanity. (Getty Images)
Let us never speak of these again.
Class Clown: JaVale McGee
Runs the other direction, denies things he's said into tape recorders, goes for triple-doubles in blowouts, makes ridiculous goaltends and has somehow failed to get better with John Wall on his team. McGee is funny. But someone really needs to get into his head and straighten things out or a world of potential is going to be lost.
Projected Defensive Player of the Year: Luol Deng
Andre Iguodala deserves it, but Deng may end up winning it simply based on reputation. If Deng is healthy, he's the best perimeter defender in the league. Dwight Howard has struggled at times and hasn't been as dominant. If the coaches really vote this one right, Deng should win as long as he continues to get healthy and Tom Thibodeau doesn't run him into the ground.
Smoke and Mirrors Award for False Relevance: Atlanta Hawks
Always a top-six team in the standings, never a top-six team in the conference. The Hawks have managed to survive a drop-off from Joe Johnson and the injury to Al Horford. The team is supremely in need of a complete makeover but none seems imminent. So they will continue to drift through the season, winning games and getting no credit, losing games and getting hammered, never going up or down. Consistency in this league is often heralded as success. But with the Hawks, it's always seen as evidence of either a mediocre student over-achieving or an excellent student never living up to potential.
Most Likely to Blow It Up: Boston Celtics
The holes have not been plugged. They have not rounded into shape. They have not righted the ship. The experience has not come through. The casual approach to conditioning has not yielded the results they want. They did not coast, they stumbled. They did not cruise, they crashed. They are alive only because of Rajon Rondo and the weakness of the Eastern Conference. The time has come to end this and move on. Sentimentality is not worth wasting time, because if the big free agents get re-aligned and the Celtics have nothing to move forward with, they'll be back in the stone age they were in before the Big 3 arrived.
Carmelo Anthony Award for MeloDrama: Dwight Howard
"I want a trade! But I love it here! But I want more input on decisions in the front office! But I'm just trying to play! I'm not talking about free agency! Except to talk to you about how much I'm not talking about it! I love my teammates! But I love Deron Williams! I love New York! But I love Orlando! I love my mom! But I make my own decisions!"
It's like watching a teenage girl decide between four boys while the one she's with can't figure out what he did wrong.
Projected Rookie of the Year: Kyrie Irving
Ricky Rubio's a nice story. Neat passes. Irving should have been an All-Star and is going to be an elite player in this league over the next decade. Cleveland's rebuild is on its way, just 19 months removed from "The Decision."
Most Likely to be Traded: Stephen Jackson
Steve Nash could go. Dwight Howard might go. Pau Gasol, Andrew Bynum, Josh Smith, Rajon Rondo, Jose Calderon. But Jackson is owed a lot of money, the Bucks don't need him, and he and Skiles hate each other. He's a bad fit in the locker room and a bad fit on the floor. It's .50 cents on the dollar time in Milwaukee for CapJack.
The 2012 Version of the 2011 Memphis Grizzlies:
The Houston Rockets. I'm starting to buy into Houston as a surprise team. Memphis used team play, star power from Zach Randolph and a unique matchup with San Antonio to get into the second round. Houston has a Should-Be-All-Star in Kyle Lowry and world of versatility in lineups they can deploy. Throw in the work that Kevin McHale has done and the fact they're 4-4 this year against division opponents (three of which are likely playoff teams) and you've got a great shot at Houston making a surprising, if ultimately futile, run. Hurray, another mid-first-round lottery pick!
Most Likely to Hit the Rookie Wall:
MarShon Brooks. Brooks is going to be a terrific scorer in this league for a long time. He's got an elite set of offensive skills and tremendous length. He can play in an offense, too. But his game is predicated on shooting percentages and that's the kind of thing that can drop off when it hits the rookie wall. I full expect to have jinxed Brooks and he will now go out and win Rookie of the Year. His play in the win over the Knicks last week was superb.
Most Likely to Make Himself a Name by Season's End: Paul George
Should have won the dunk contest. Played Rose great last year. The Pacers are better than last year and are primed for a playoff run. Getting them out of the postseason is going to be a serious job, and George is going to be a huge part of it. More versatile on offense, better on defense, athletic, explosive, with range. George isn't going to score 40. But he's going to be a part of a lot of playoff wins for Indiana and is going to be that guy in the playoffs that make sports bars of opposing teams groan "Not that guy again!" time and time over. By the way, I've started calling him Mega-Man because it seems like every game he picks up a new ability he didn't have before.


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Sugar-Free and Gluten-Free Cookies with Almond and Flaxseed Meal
|Sugar-Free and Gluten-Free Cookies with Almond and Flaxseed Meal
It's President's Day weekend in the U.S., but before I take a few days off, I thought I'd share this cookie recipe I made recently. If you've looked through my recipe archives, you see I have a few Low-Sugar Desserts, although I don't make a lot of cookies. However, a few years ago I had a reader named Adam who inspired me to post a recipe for low-carb snickerdoodles. That recipe uses a whole stick of butter and although the cookies were delicious and it worked for Adam, who was an Atkins dieter, it's not something I would make now.
Sugar-Free and Gluten-Free Cookies with Almond and Flaxseed Meal (Gluten-Free)
(Makes about 15 cookies, adapted from RecipeZaar.)
1/2 cup trans-fat free margarine (be sure not to choose a margarine that has water for the first ingredient)
1 1/2 cups almond meal (I use Bob's Red Mill brand)
2 T flaxseed meal (I use Bob's Red Mill brand)
1 cup granular Splenda or Stevia-in-the-Raw Granulated Sweetener
1 egg
1/2 tsp. vanilla (I use Mexican vanilla)
1/4 tsp. baking soda
1/4 tsp. cream of tartar
Put on a plate for rolling cookies:
2 T Splenda or Stevia-in-the-Raw Granulated Sweetener
1 tsp. ground cinnamon
Preheat oven to 350 F. Combine 2 T Splenda and ground cinnamon on plate. Use a tablespoon to scoop out dough, then roll into a small ball and roll in Splenda or Stevia/cinnamon mixture. Place on cookie sheet about 2 inches apart. (I used a fork to smash the cookies down, which is optional.)
Bake for 15-20 minutes, until cookies are slightly firm and starting to brown. (I got distracted and baked them a few minutes too long, but they still tasted great.) Cookies will get more firm as they cool. These cookies are a bit more crumbly than regular cookies, but I thought the crisp texture was really nice. Cookies will keep for a few days, or can be frozen for a few months.
Printer Friendly Recipe
South Beach Suggestions:
Nutritional Information?
I chose the South Beach Diet to manage my weight partly so I wouldn't have to count calories, carbs, points, or fat grams, but if you want nutritional information for a recipe, I recommend entering the recipe into Calorie Count, which will calculate it for you.
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Revision Information
- Reviewer: Michael Woods, MD
- Review Date: 04/2016 -
- Update Date: 04/13/2016 -
1717 Pennsylvania Avenue NW
Suite 1025
Washington, DC 20006
This website provides a variety of materials discussing sleep apnea, including a brochure, a phone app, and many other educational materials. You can participate in the "Sleep Apnea Support Forum" and post questions about the condition and treatments.
PO Box 30105
Bethesda, MD 20824-0105
NHLBI provides fact sheets on sleep apnea and other sleep disorders, as well as heart, lung, and blood disorders. Look here for information on current areas of research, clinical trials seeking subjects, meetings and conferences, and a link to the organization's sister website, the National Center on Sleep Disorders Research (NCSDR).
1010 North Glebe Road
Suite 420
Arlington, VA 22201
The National Sleep Foundation (NSF) offers brochures, newsletters, and reports on sleep apnea and other sleep disorders. The website features a list of related links and an area called the NSF Community Sleep Awareness Partners (CSAP), where you can find sleep disorder specialists in your area.
1650 Diagonal Road
Alexandria, VA 22314-3357
The American Academy of Otolaryngology—Head and Neck Surgery (AAO-HNS) is the world's largest organization representing specialists who treat the ear, nose, throat, and related structures of the head and neck. On this website, you can find newsletters, reports about obstructive sleep apnea and other sleep disorders, and a lengthy list of links to related websites.


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Money is vital for modern democracies. Without it, citizens would not have the means to convey ideas nor compete for political power through electoral processes. However, money can distort the democratic ideals of fair competition through unbalanced access to resources to some individuals or political groups, upsetting one of the cornerstones of democracy – the concept of 'one person one vote'.
Explore this Theme Page to, among other things:
U4 recently attended a UNDP event that brought together Supreme Audit Institutions from different countries in the Balkans to discuss their role in monitoring money financing political parties and electoral campaigns. The negative influence of money in these processes, compounded with a necessity for resources to allow information to reach out to voters, is a problem to be addressed. To learn more about different actors and their roles in monitoring how money influences political processes, take a look at this U4 brief: Political finance: State control and civil society monitoring.


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Recession-Driven Violence? "We are seeing more people at the shelter than we ever have before," Kristin Carmichael, manager at the Esperanza Shelter for Battered Families, says. "I really think that folks who used to hang on by their fingernails—to housing or by getting transportation out of town or to living with family members—they're seeking shelter."
The 12 months ending in April saw a 56 percent increase in demand at the shelter over the previous year. The 45-bed shelter measures demand by "days of care" provided. In that period, the days of care spiked to 10,500 from 6,700.
"Summer is our absolute busiest time, and we're coming into that time—so we could see even higher numbers," Carmichael says. "Shelters statewide are seeing this increase, happening around the same time the economy took a dive." Reach the shelter's toll-free crisis line at 800-473-5220. To volunteer, call 505-473-5200.
Broker Battle: As the Santa Fe real estate market sours, so do friendships. Such is the story behind a recent lawsuit filed by Pamela Nittolo, an associate broker at Santa Fe Realty Partners. Nittolo worked for the California-based Shaklee Corporation, a "multi-level marketing" company, before moving to Santa Fe and getting into the real estate business. Her complaint, filed late last month in 1st Judicial District Court, says she paid $36,600 toward the purchase of a condo on W. Santa Fe Avenue in 2007, but was then shut out of her role in the property's management by her partners, Prudential Santa Fe Real Estate broker Barbara Millstein and her husband, Harry Abramson. Nittolo claims Millstein and Abramson recently listed the property for sale "unilaterally," intending to deprive her of a commission on the sale. Millstein says that's news to her. "We were friends and we purchased a condo together as an investment. And that was it. And now we're looking at selling it and dividing up the profits," Millstein says. "If there's a suit, it's a frivolous suit, and there really is no merit to it." She says renters now live in the condo, but don't pay enough to cover the mortgage.
Books Before Bikes: This sad literacy news from City of Santa Fe spokeswoman Laura Banish suggests some SFR readers aren't reading too carefully. Banish, who organized Bike to Work Week (happening now!) for the city, placed an ad in last week's paper promising a chance to win prizes for anyone who found the Bike to Work Week logo hidden elsewhere in that edition. The ad ran on page seven. Banish says a shocking number of people responded they'd found the hidden logo…in the ad on page seven. Wrong answer, folks. Do Banish and all Santa Fe cyclists a favor by taking the city's new commuter survey, which you can find on our blog, SFReeper.com.


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Federal Emergency Management Agency
1 Jul 2012 at 17:33
When getting prepared Kingpenwell0829 reminds you to bring things like any medicine you may need and eye glasses to be able to see.
With Kingpenwell0829
Press | FAQ | Terms of Agreement | Privacy Policy | Copyright © 2011 Flatter World Inc.
FLAT STANLEY® is a trademark of the Trust u/w/o Richard C. Brown f/b/o Duncan Brown. Used with permission. All rights reserved


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There's a surprise with this recap because I've finally succumbed to peer pressure and done a recap with photos. Yes, I know that some of you love the photos! This was something that was out of the question time-wise when I was doing the recap more frequently. It's time consuming to copy, re-size, and optimize this many photos, and I've never been able to decide whether I think the photos are an incentive or a disincentive to click through to the original post, so I'm not sure still if I'll do it every time! With so many photos, I've optimized them so the post won't be slow to load, so click through to the site to see them as they were done by the photographer. One thing that's no surprise at all is that there's a magnificent array of interesting dishes again this week, so let's move on and talk about the food.
Arbi (Taro)
Arugula, Chickpeas
Arugula, Apples
Basil, Assorted Veggies
Basil, Thyme, Saffron
Beetroot, Mint
Black Beluga Lentils
Blueberries, Greens
Buckwheat, Potatoes, Parsley
Chard, Greens, Chickpeas
Chives, Parsley, Sage
Fennel, Apple, Celery
Garlic Chives
Ginger, Bok Choy, Napa Cabbage
Ginger, Broccoli
Japanese Mountain Yam
Leeks, Assorted Herbs
Lima Beans, Peas
Meyer Lemons
Mizuna Mustard Greens
Parsley, Golden Raisins
Potatoes, Mango, Ginger
Red Rice, Jasmine
Ridgegourd and Toor Dhall
Rye Flour, Assorted Herbs
Spinach, Lemon, Green Garlic
Sprouted Wheat Flour
Star Anise, Cilantro, Basil, Lime
Tomatoes, Red Bell Peppers, Garbanzos, Artichokes
Thyme, Leeks, Spinach, Mushrooms
Watercress, Pao Shen, and Wolfberries


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daverupa wrote:Indeed, unremitting satipatthana is a better goal to set than jhana, when first beginning samadhi.
Mojo wrote:I see them as my gateway into going further down formal Anapanasati practice that culminates with vipassana.
Mojo wrote:I'm not asking about piti and sukkha because I want to experience them for the sake of the experience but because I see them as my gateway into going further down formal Anapanasati practice that culminates with vipassana.
In the first steps of this practice, those concerned with the kaya (body), we study the breath in a special way. We note every kind of breath that occurs and study what each is like. Long breaths, short breaths, calm breaths, violent breaths, fast breaths, and slow breaths: we must know them all. Of all the different kinds of breath which arise, know what nature each one has, know its characteristics, and know its functions.
Observe what influence the different breaths have upon the flesh-body. The breath has a great influence on the rest of the physical body and this influence needs to be seen clearly. Observe both sides of the relationship until it is obvious that they are interconnected and inseparable. See that the breath-body conditions and concocts the flesh-body.
We must learn how to observe in more detail, that is, to observe the reaction or influence of the different kinds of breathing. What reactions do they cause, how do they influence our awareness? For example, when the breathing is long, how does it influence our awareness. What reactions does the short breathing cause? What are the influences of coarse and fine breathing, comfortable and uncomfortable breathing? We observe the different types of breath and their different influences until we can distinguish clearly how the long and short breaths, coarse and fine breaths, and comfortable uncomfortable breaths differ . We must know the variations in the reactions to and influences of these various properties of the breath, of these qualities that influence our awareness, our sensitivity, our mind. (52)
Along with the above observations, we need to watch the effect or flavor of the different kinds of breath. The flavors that arise are kinds of feelings, such as, happiness, non-happiness, dukkha, annoyance, and contentment. Observe and experience the flavors or effects caused, especially, by the long breath and short breath, by the coarse breath and fine breath, and by the easy breath and uneasy breath. Find out how it is they have different flavors. For instance, we will see that the long breath gives a greater sense of peace and well being, it has a happier taste than the short breath. Different kinds of breath bring different kinds of happiness. We learn to analyze and distinguish the different flavors that come with the different kinds of breath that we have scrutinized. (53)
Finally, we will discover the various causes that make the breath either long or short. We gradually will find this out for and by ourselves. What causes the breathing to be long? What kind of mood makes the breath long? What kind of mood makes it short? Thus, we come to know the causes and conditions that make the breath long or short.
The body which is the causal conditioner is given the name kaya-sankhara (body conditioner) to distinguish it from the other, the one effected by the conditioning, the "conditioned body." Work on this fact in the mind, seeing it as if it were physically tangible. See the one group condition and nurture the other. See them arise together, fall together, coarsen together, become fine together, grow comfortable together, and become uncomfortable together. Realize how intimately they are connected.
This is what is meant by "seeing all bodies," Watch both bodies together and see them condition each other. This is valuable for seeing truth more extensively, for realizing anatta, even. In seeing this interrelationship, we see that what occurs is merely a natural process of conditioning. There is no atta, no self, no soul, no such thing at all involved. Such understanding can have the highest benefit, although it may be somewhat beyond the specific object of this step. For now, however, we only need to understand this fact of conditioning enough to be able to regulate the flesh-body, to calm it by regulating the breath-body.
Zakattack wrote:Every tetrad (satipatthana) of Anapanasati includes vipassana.... The 4th tetrad is full scale vipassana, where impermanence itself is predominant.
daverupa wrote:Samatha and vipassana are paired qualities which interact with satipatthana, per SN 35.204.
porpoise wrote:daverupa wrote:Samatha and vipassana are paired qualities which interact with satipatthana, per SN 35.204.
An interesting sutta, but I'm not sure I understand the relationship being described here between samatha and vipassana ( the swift pair of messengers ), mindfulness ( the gatekeeper ) and consciousness ( the commander ) - see the extract below. It sounds as if consciousness is primary, with mindfulness relegated to the role of giving directions.
"A swift pair of messengers, coming from the east, would say to the gatekeeper, 'Where, my good man, is the commander of this fortress?' He would say, 'There he is, sirs, sitting in the central square.' The swift pair of messengers, delivering their accurate report to the commander of the fortress, would then go back by the route by which they had come.
The message is this:
The fortress stands for this body... The gatekeeper stands for mindfulness. The swift pair of messengers stands for tranquillity (samatha) and insight (vipassana). The commander of the fortress stands for consciousness."
porpoise wrote:The text of the 4th tetrad is clearly describing vipassana ( anicca ), but could you say which text lines in the first 3 tetrads are explicitly describing vipassana? Phrases like "sensitive to feeling" and "experiencing the mind" seem more like sati than vipassana.
daverupa wrote:The important point is to recognize that they develop within satipatthana practice. You had said that anapanasati culminates in vipassana, but in fact anapanasati fulfills satipatthana - both samatha and vipassana ought already to have begun development prior.
Zakattack wrote:For practicality, I would suggest to view Anapanasati as: 1st tetrad: 75% samatha 25% vipassana; 2nd tetrad: 50% samatha 50% vipassana; 3rd tetrad 25% samatha 75% vipassana; 4th tetrad 100% vipassana. As for the Pali, the same word 'paṭisaṃvedī' (experiencing; sensitive to) is used in the 1st, 2nd & 3th tetrads.
porpoise wrote:IMO some interpretations of the 4 tetrads are basically just describing satipatthana practice, which seems to me missing the point.
"And how is mindfulness of in-&-out breathing developed & pursued so as to bring the four frames of reference to their culmination?
"And how are the four frames of reference developed & pursued so as to bring the seven factors for awakening to their culmination?
both samatha and vipassana ought already to have begun development prior [to anapanasati]
how a monk develops mindfulness immersed in the body.
Kamran wrote:Thanissaro is an excellent choice in my opinion due to the daily talks he provides that address problems you encounter in real-life meditation practice. Instructions only go so far; you have to figure things out yourself by practicing.
You may want to explore his collection of talks that accompany his "With Each and Every Breath" manual.
http://www.dhammatalks.org/mp3_collecti ... verybreath
frank k wrote:In the Chinese Agama parallel to anapana 16 steps, they are much more explicit in step 3 of anapanassati, "sabba kaaya patisamvedi", "experiencing the whole [anatomical] body".
Kāyesu kāyaññatarāhaṃ, bhikkhave, evaṃ vadāmi yadidaṃ – assāsapassāsā.
I tell you, monks, that this — the in-&-out breath — is classed as a body among bodies.
So if a monk should wish: 'May neither my [anatomical] body be fatigued nor my eyes, and may my mind, through lack of clinging/sustenance, be released from fermentations,' then he should attend carefully to this same concentration through mindfulness of in-&-out breathing.
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Related stories: Local group alleges third Chinese maternity hotel operation in Chino Hills | Chino Hills officials caution residents | Dozens turn out to discuss Chino Hills maternity hotel | Alleged maternity hotel operation prompts criminal complaint against homeowner | Chino Hills home small part of birth tourism industry | 70 protest alleged 'maternal hotel' in Chino Hills | Chino Hills residents raise concerns about possibly of maternity hotel operation
The discovery of at least two "maternity hotels" in Chino Hills has called new attention to the controversial – but legal – phenomenon of maternity tourism.
In Chino Hills, a large house at 15250 Woodglen Drive is the alleged site of a maternity hotel for Chinese nationals seeking to give birth in the United States and obtain American citizenship for their children.
A nearby apartment complex is also a suspected maternity hotel, and there may be one or more additional sites in the city.
The Chino Hills cases are not unique. Other maternity hotels have been found locally in the San Gabriel Valley and as far as New York.
It is difficult to gauge exactly how widespread the semi-underground practice is, but foreign language websites indicate that maternity hotel operators charge several thousands of dollars for the opportunity to have a child born on American soil.
Some experts say that maternity tourism is a multi-million dollar business in the United States.
"The people who are coming abroad are people who can afford to travel," said Neha Singh, professor at Collins College of Hospitality and Management at Cal Poly Pomona. "These are not people who are the lower strata in their home countries, but still they would like to have possibility in the future for their children."
Conservative author and columnist Jerome Corsi said his studies of maternity tourism show mothers may pay as much as $50,000 to $100,000 for the medical care and accommodations.
"If your net worth is in the multi-millions, this may be a reasonable price to pay to be a U.S. citizen," Corsi said. "It is not for the average person. And that's what you have to distinguish, this is high-end opportunities for people who have money."
Singh said Corsi's estimate is too high. She pegged the likely costs as being closer to $25,000.
The practice of birth tourism is controversial because American birthright citizenship laws allow children born under such circumstances to obtain U.S. citizenship despite being brought up in another country.
Birthright citizenship is established under the 14th Amendment, which states that people born under the jurisdiction of the United States receive citizenship.
The amendment was ratified in 1868 to protect the rights of former slaves after the Civil War. The Supreme Court has affirmed that it grants citizenship to the children of foreign nationals.
Some immigration conservatives say the 14th Amendment is not meant to apply to the children of tourists who despite being born in the United States, would be raised in another country.
"Somebody who is just here for the purposes of giving birth has that allegiance to another country," said Ira Mehlman, spokesman for the Federation for American Immigration Reform.
Current law, however, makes it possible for a relatively small but growing number of maternity tourists to obtain citizenship for their newborns.
That opportunity for citizenship seems to be creating a business opportunity for those who facilitate maternity tourists' travels.
For example, a translated version of the Chinese-language website www.jiiaa.com refers to an organization called "Mabel House" with a headquarters in Shanghai. The website refers to several hospitals in and around the San Gabriel Valley where Chinese-speaking doctors are available.
The website states that hospitals are identified only for reference purposes. The spokesman for Garfield Medical Center, which in Monterey Park is one of the listed hospitals, said the medical center does not promote birth tourism and has already asked to have its name removed from another website, asianm.com.
That website was offline on Friday after being operative earlier in the week.
Garfield Medical Center spokesman Erik Jiang said the hospital does not place overseas advertisements to encourage birth tourism and does not deny care on the basis of a patient's immigration status or nationality.
Another Chinese language website – usanybaby.com – advertises medical delivery and one month's stay in New York for mothers. Listed charges can add up to $24,000.
The website also advises mothers that a baby born in the United States will obtain American citizenship and be able to attend public schools and permanent residency.
"If you want to give the baby a good education, life, environment, please move fast," the website advises.
That website's operators did not return calls for comment.
A May 2011 NBC New York report quotes a person named Anna as the owner of the house advertised on usanybaby.com.
Anna said in that report that she rents to women who can enter the United States legally on a visa and makes the necessary medical arrangements. Anna advises her customers to travel before their pregnancy is obvious to anyone who sees them.
The pricetag to stay at the facility ranges from $2,000 to $4,800. This includes three meals a day, supplies, a twice-weekly shopping shuttle, an on-call nanny and more.
Birth tourism attracts tourism from countries such as China, Taiwan, South Korea and India, Singh said.
Turkey is also believed to be a country where a birth tourism industry has become established.
There are no exact numbers that show exactly how many mothers travel to the United States to obtain citizenship for their children.
Available statistics show that 7,719 births to non-resident mothers were recorded in the United States. That's a small number compared to the nearly 4 million births recorded that year, but 54 percent greater than the number of births to non-resident mothers in 2000.
If these numbers indeed reflect a rise in maternity tourism, it could be because entrepreneurs from around the world found an opportunity to provide travel services to families who do not have relatives in the United States who can help their children obtain citizenship.
"So initially, it could be individuals trying to get help from friends or family members to help them achieve this, and over time you have these people who do not have those connections but still want to be able to have a child who is natural born citizen," UC Riverside political scientist Karthick Ramakrishnan said.
"That's when you see enterprises pop up when people cannot just rely on family networks."
The 7,719 births to non-residents in 2010 are also a small number when compared to the more than one million immigrants who achieved legal residency that year.
"It's not typical of the Chinese or Asian-American experience. Their families are here and they intend to stay here," said Karin Wang, vice president of programs and communications for the Asian Pacific American Legal Center.
"I would say maternity tourism is an aberration," she added.
Wayne Austin, the former president of the San Bernardino Convention and Visitors Bureau, said he has never heard of any hotel companies incorporating birth tourism into their business models.
"I've been in this business for 30-some years and this is nothing that's come across my desk," said Austin, who is now general manager for Arrowhead Country Club in San Bernardino.
Given that maternity tourism is a semi-underground business, it's difficult to establish exactly how much money is to made by those participating in it.
"I'm just guessing, but yes, I'd say it's a multimillion industry and business," Corsi said. "But it's a specialty business. It's not going to show up in terms of being monitored or measured among travel agents. It's going to be off the charts."
Reach Andrew via email, call him at 909-483-8550, or find him on Twitter @InlandBizz.
Reach Canan via email, call her at 909-987-6397 ext. 425, or find her on Twitter @ChinoValleyNow.


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