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This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
The virologic synapse (VS), which is formed between a virus-infected and uninfected cell, plays a central role in the transmission of certain viruses, such as HIV and HTLV-1. During VS formation, HTLV-1-infected T-cells polarize cellular and viral proteins toward the uninfected T-cell. This polarization resembles anterior-posterior cell polarity induced by immunological synapse (IS) formation, which is more extensively characterized than VS formation and occurs when a T-cell interacts with an antigen-presenting cell. One measure of cell polarity induced by both IS or VS formation is the repositioning of the microtubule organizing center (MTOC) relative to the contact point with the interacting cell. Here we describe an automated, high throughput system to score repositioning of the MTOC and thereby cell polarity establishment. The method rapidly and accurately calculates the angle between the MTOC and the IS for thousands of cells. We also show that the system can be adapted to score anterior-posterior polarity establishment of epithelial cells. This general approach represents a significant advancement over manual cell polarity scoring, which is subject to experimenter bias and requires more time and effort to evaluate large numbers of cells.
Anterior-posterior (AP) cell polarity is defined as the asymmetric distribution of macromolecules and organelles in the x-y cell plane. This process, which becomes initiated by localized activation of receptors at the cell surface, is essential for diverse functions, including embryonic morphogenesis, tissue repair, and immune surveillance [
The immunological synapse (IS) is a specialized cell-cell contact membrane junction formed by the interaction between a T-cell and antigen-presenting cell. Formation of the IS triggers activation of cell signaling cascades that promote AP polarization and contribute to activation, survival, and migration of T-cells [
Previous studies have scored AP cell polarity establishment in microscopic images using a time consuming and laborious manual process [
To induce IS formation and AP polarity establishment, we chose the established method of incubating human Jurkat T-cells with anti-CD3/CD28 coated beads [
In the first step of the automated system, confocal microscopy images are collected and compiled by a Pipeline Pilot algorithm. The algorithm determines the eligibility of each cell for MTOC:bead angle determination based on two strict criteria. (1) The cell must contact only one bead and must not contact another cell to ensure that the cell receives only one polarization signal. (2) The cell must contain only one MTOC. Cells meeting both criteria are subjected to the second step of the automated system, in which a reference line, designated the 0° line, is drawn between the center of the nucleus and the center of the bead attached to this cell (
To assess cell polarity establishment, MTOC:bead angles calculated for cell sample populations are compiled and sorted into four different 90° regions: region 1 includes two adjacent 45° sectors bisected by the 0° line; region 2 includes two separate 45° sectors adjacent to and on either side of region 1; region 3 includes two separate 45° sectors adjacent to and on either side of region 2; region 4 includes two adjacent 45° sectors next to region 3 (
We first used the automated system to establish the kinetics of AP polarity establishment following incubation of Jurkat cells with anti-CD3/CD28 beads for 5, 10, 15, 20, 30, 60 or 120 min (
Automated MTOC:bead angle determinations. (
Anti-CD3/CD28 beads rapidly induce T-cell polarization. Jurkat T cells were incubated with anti-CD3/CD28 beads for 5, 10, 15, 20, 30, 60, or 120 min, stained with DAPI and for pericentrin, and subjected to automated imaging and MTOC:bead angle determinations. The percentage of each cell sample population that sorted into regions 1–4 is shown. The number of scored cells (n) is shown in the legend for each time point. The horizontal dashed line indicates the 25% random cell distribution within each region expected for unpolarized cells. **, p < 0.007; ***, p < 0.001.
We next investigated whether grouping cells in smaller or larger sized regions would yield additional information about cell polarity. For this purpose, we sorted data from a new 20 min time point experiment, as well as re-sorted
High resolution analysis of MTOC position during T-cell polarization. Data from the 20 min time point in
As a control, we also asked whether anti-CD3/CD28 beads specifically promote polarity establishment in Jurkat T-cells. This was accomplished by incubating Jurkat cells for 20 min with negative control anti-CD19 coated beads, which do not bind to or elicit IS formation or polarity establishment in Jurkat cells [
To determine whether our high throughput imaging system could be applied to systems other than Jurkat T-cells, we next adapted the automated angle calculation system to score AP polarity establishment in epithelial cells. In human MCF-10A mammary epithelial cells, cell surface receptors initiate signaling cascades in response to a polarizing stimulus, such as wounding of a cell monolayer. In wound-edge cells, this stimulus promotes forward microtubule polarization toward the wound and rearward reorientation of the Golgi apparatus and MTOC behind the nucleus. Thus, as for Jurkat cells, we used the MTOC as a marker to score polarity establishment, which was determined by the MTOC distribution within three 120° regions (regions 1–3) of wound-edge MCF-10A cells (
To test the automated system on MCF-10A cells, we overnight EGF-starved a confluent cell monolayer, which was then scratch-wounded multiple times with a P200 pipette tip. Negative control cells were fixed immediately post-wounding (t = 0 h), whereas experimental cells were incubated in complete medium for 6 h post wounding to allow for cell polarization and then fixed. Cells were stained with anti-pericentrin antibody and DAPI, and images of cell fields were collected manually. In cell images, Adobe Photoshop was used visually to place a red dot in the cytoplasm between the center of the nucleus and the center of leading edge membrane. The automated system was employed (1) to connect the center of the red dot to the center of the nucleus, thereby creating the reference 0° line, (2) to connect the center of the MTOC to the center of the nucleus, thereby creating the MTOC line, and (3) to calculate the smallest MTOC:leading edge angle formed by the intersection of the 0° and MTOC lines (
Anti-CD19 beads fail to promote T-cell polarization. Jurkat cells were incubated with negative control anti-CD19 beads or positive control anti-CD3/CD28 beads for 20 min, and stained with anti-pericentrin antibody and DAPI. High throughput image acquisition and angle analysis was performed. The percentage of cells containing an MTOC in the indicated region is shown. The graph represents three independent experiments. The number of scored cells (n) is shown in legend. The horizontal dashed line indicates the 25% random cell distribution within each region expected for unpolarized cells. *, p < 0.05; **, p < 0.002.
As an additional negative control for cell polarity establishment, we similarly scored images of internal monolayer cells stained for pericentrin. In this case, however, an artificial wound was introduced by using Adobe Photoshop to draw a straight arbitrary line (yellow) onto images of unwounded cell monolayers. The automated scoring system was then used to calculate the smallest angle formed between the 0° line (red), which is perpendicular to the artificial wound edge and runs through the center of nucleus, and the MTOC line (green), which connects the center of the MTOC to the center of the nucleus (
Use of the automated system to score AP polarity establishment in human MCF‑10A mammary epithelial cells. (
Results in
As was done with Jurkat T-cells in
We described the development of a new automated, high throughput method to score AP cell polarity establishment by capturing cell images while excluding abnormal cells using a strict set of criteria, calculating precise MTOC:bead or MTOC:leading edge angles, and compiling the data. In addition, flexibility of the approach allowed us to analyze cells of different types and to sort them into cell regions of various sizes. The system also facilitated data collection for thousands of cells. These key features permit detection of statistically significant differences even between small cell regions, providing high-resolution insights into the mechanisms of cell polarity establishment.
The versatility of our algorithm also allowed us to test a detailed hypothesis. We initially sorted Jurkat T‑cell populations into four 90° regions. In this case, unpolarized cells should distribute approximately equally (25% of cells/region) within each region, whereas polarized cells should instead distribute predominantly in the region encompassing the polarization stimulus (anti-CD3/CD28 coated bead contact site). Indeed, in Jurkat cells, we observed a significantly increased accumulation of MTOC in regions adjacent to the site of bead contact, an effect that was maximal after a 20 min incubation period. This finding led us to hypothesize that the MTOC migrates relatively rapidly to a position immediately adjacent to the cell polarization stimulus. We investigated this idea by re-sorting cells into smaller 20° or 30° regions. To our surprise, we found that the MTOC does not concentrate closest to the bead contact site, but rather to a 20–30° region on either side of this site (region 3 in
To determine whether the automated system could be adapted to other cell types, we modified the algorithm to score polarity in adherent MCF-10A epithelial cells. At present, a reference point is manually added to the images of epithelial cells. To fully automate polarity scoring of migrating epithelial cells, we plan in the future to use an antibody to mark the leading edge membrane of wound edge cells and to develop an algorithm that places a dot at the center of the leading edge membrane. We succeeded in scoring polarity from large numbers of epithelial cells. Furthermore, contrary to polarized Jurkat T cells, the MTOC of polarized MCF-10A cells is known to concentrate in the region directly opposite from the polarizing stimulus. Indeed, we observed maximal MTOC accumulation in the region directly opposite from the polarizing stimulus, rather than offset from the predicted pattern seen in Jurkat cells. The difference between MTOC accumulation patterns in Jurkat and MCF-10A cells may depend on the physical nature of the stimulus itself as no equivalent to the bead-induced dimple in Jurkat T cells is seen in scratch-wounded MCF‑10A epithelial cells. It is also possible that MTOC migration away from the polarizing stimulus in MCF-10A cells does not enforce the same physical restriction for MTOC movement postulated in Jurkat cells.
The new automated system also has important virological applications. The loss of cell polarity is a feature of epithelial-derived cancer cells, and there is increasing evidence that this defect in polarity may play a pivotal role in the pathogenicity of cancer [
Human Jurkat T cells and MCF-10A human mammary epithelial cells were acquired from the American Type Culture Collection (Manassas, VA, USA). Jurkat cells were maintained in RPMI 1640 media with L-glutamine containing 10% fetal bovine serum (FBS) and antibiotic-antimycotic solution (1,000 units penicillin, 1,000 µg streptomycin, and 0.25 µg amphotericin B per mL), whereas MCF‑10A human mammary epithelial cells were maintained in Dulbecco's Modified Eagle Medium:F12 supplemented with 5% horse serum, 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, and 20 µg/mL gentamicin. All cells were cultured at 37 °C under a 5% CO2 atmosphere.
Exponentially growing Jurkat cells and Dynabeads conjugated to anti-CD3/CD28 (Invitrogen Dynal AS, Oslo, Norway, catalog # 111.31D) or anti-CD19 (catalog # 111.43D) antibodies were separately washed in PBS (Invitrogen catalog # 14190-144) containing 0.5% FBS (PBS FBS buffer). Washed Jurkat cells (8 × 104) mixed with beads (4 × 104) in 0.1 ml total volume of PBS FBS buffer were added to each poly-L-lysine-coated well of a 96-well optical grade plate (Greiner Bio-One, Monroe, North Carolina, catalog # 655892P) and allowed to settle for 20 min at 37 °C in a tissue culture incubator. After aspiration of the PBS-FBS buffer, cells were fixed in 3.5% paraformaldehyde (PFA) in PBS for 10 min at room temperature (RT), rinsed, and permeabilized with 0.1% Triton-X 100 in PEM buffer for 30 min at RT. Permeabilized cells were washed three times for 5 min, blocked in filtered IF buffer containing 10% goat serum for 1 h at RT, and incubated with rabbit anti-pericentrin (1:1000) (Abcam, Cambridge, MA, catalog # ab4448) in KB buffer for 1 h at 37 °C in a tissue culture incubator. After incubation with the primary antibody, cells were washed three times for 5 min, incubated with goat anti-rabbit AlexaFluor-488 (1:5000) (Invitrogen, Carlsbad, California catalog # A-11008) in KB buffer for 45 min at 37 °C in a tissue culture incubator, washed three times for 5 min, counterstained with 1 µg/µL DAPI, and rinsed with PBS. Unless otherwise indicated, rinses and washes mentioned above were done with PEM buffer (80 mM PIPES, pH 6.8, 5mM EGTA, pH 7.0, and 2 mM MgCl2). Images were collected on a Beckman IC100 high throughput microscope at the Baylor College of Medicine Integrated Microscopy Core.
MCF-10A cells were seeded at 3 × 105 cells/well on 2-well glass slides in complete medium and, upon reaching confluency, were incubated overnight in complete medium lacking EGF. The cell monolayer was then scratch-wounded five times per well using a P200 pipette tip, washed with complete medium lacking EGF to remove detached cells, and incubated in complete media for 6 h. Immunofluorescence assays were conducted as described for Jurkat cells, except PFA was quenched with 7.5% glycine solution prior to blocking with goat serum, goat anti-rabbit AlexaFluor-488 (1:500) (Invitrogen, Carlsbad, California catalog # A-11008) secondary antibody was used, and images were captured on a Zeiss Axioplan upright microscope or a Nikon A1 confocal microscope at the Baylor College of Medicine Integrated Microscopy Core.
Background subtracted from images was approximated as m + m1/2, where m is the minimum pixel intensity of the image. Objects (sets of contiguous foreground pixels) in different channels were detected using a local threshold approach in which an image pixel is placed in the foreground if its intensity is greater than the mean intensity in a surrounding window plus some constant, C, which was empirically assigned based on its ability to exclude non-objects and retain real objects. Holes in objects were filled to make them solid. Any objects touching the border of an image field were excluded. Nuclei were detected in the DAPI channel using a window of 11 × 11 pixels and C = 3; MTOC were detected in the GFP channel using a window of 7 × 7 pixels and C = 10; and beads were detected in the red channel using a window of 7 × 7 pixels and C = 20.
Cell boundaries were approximated from the nuclear objects using a distance map, whose pixels have values based on their distance in pixels to the nearest nuclear foreground pixel. Contiguous pixels in the distance map with a value of five or less were used to define cell objects. Beads that did not overlap with cells were excluded. Next, the centers of all objects in an image field were calculated, and these measurements were used to associate a cell with the closest bead and MTOC. Cells associated with more than one bead were also excluded. For the remaining cells, the MTOC:bead angle, defined as the smallest angle between a line from the center of the nucleus to the center of the bead (0° reference line) and a line from the center of the nucleus to the center of the MTOC (MTOC line), was calculated. Automated image preprocessing, segmentation, filtering, and measurement were performed in Pipeline Pilot 8.0 using a custom-built protocol. The custom code for the Pipeline Pilot software (Accelrys) used to score T-cell and epithelial cell polarity will be provided upon request through a Materials Transfer Agreement (MTA) between the end-user institution and the Integrated Microscopy Core of Baylor College of Medicine.
The above protocol was modified slightly to process adherent epithelial cells, which were present at a higher density than Jurkat cells. Nuclear objects from the DAPI channel were detected using a local threshold with a window size of 60 × 60 pixels and C = 5. To identify overlapping nuclei, region splitting was applied to the objects by calculating an inverse distance transform on the thresholded image (that is, measuring a pixel's distance to the nearest non-foreground pixel) and then finding peaks in the resulting distance map, where each peak corresponds to a nuclear seed. Starting from these seeds, a watershed transformation was run on the distance map to identify overlapping nuclei, which were discarded from the analysis using a shape filter. MTOC were detected as described for Jurkat cells. Leading edge cells were located visually from the DIC channel and then labeled manually with a red dot to mark the center of the leading edge. The centers of the nucleus, MTOC, and leading edge were calculated and used to determine the MTOC:leading edge angle.
Angles generated by the algorithm in Pipeline Pilot were sorted and analyzed in Microsoft Excel.
ANOVA with Tukey post-hoc analysis using R statistical software was used.
This paper describes a new automated, high throughput system to score cell polarity establishment by capturing cell images, applying strict criteria to exclude unwanted cell images, compiling data, and calculating MTOC:bead angles. This flexible system permits sorting and evaluation of MTOC positions within cells using a variety of cell sectoring strategies. The system rapidly analyzes thousands of cells, facilitating acquisition of statistically significant results from very small regions of the cell and high resolution insight into the establishment of cell polarity. The approach has a variety of applications, including visualization and analysis of the virologic synapse and/or the role and effect of viral oncoproteins on polarity, and can be adapted for use in other systems and cell types.
This study was supported, in part, by the United States Public Service Grant R01 CA55684 from the National Cancer Institute, National Institutes of Health, awarded to S.J.M., and by the United States Public Service Grant R01 CA058541 from the National Cancer Institute, National Institutes of Health, awarded to R.T.J. C.V.I-W. was supported by IRACDA training grant K12 GM84897 from the National Institutes of Health and K.K. was supported by the Baylor College of Medicine Comprehensive Cancer Training Program, CPRIT #RP101499. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank members of the Marriott and Javier laboratories for helpful suggestions and editorial comments. We also thank James Brougham and Debra Townley of the BCM Integrated Microscopy Core, which is supported by the Specialized Cooperative Centers Program in Reproduction U54 HD-007495 (to B.W. O'Malley), P30 DK-56338 (to M.K. Estes), P30 CA-125123 (to C.K. Osborne), and the Dan L. Duncan Cancer Center of Baylor College of Medicine. We also thank Susan Hilenbeck (BCM) for assistance with the statistical analyses.
The authors declare no conflict of interest.


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Cashew and Herb Turkey Stuffing
Recipe by Chef floWer
I was cooking a whole Turkey and I didn't know what to use for the stuffing. So my workmate suggested this recipe. She found it in a Christmas book called 'Family Circle, The tastes of Christmas, step-by-step'
Top Review by Andi of Longmeadow
I had a little chicken waiting to be roasted an stuffed with the unique and delicious herb stuffing. I love stuffing turkey or chicken, and this stuffing recipe is no exception to enhancing the same old roasted chicken. The only change I made for this recipe was using white rice instead of brown, but other then that, followed this exactly. What an incrediable taste sensation! I continue to have an abundance of fresh mint growing and that taste, coupled with the lemon zest, cashews was beyond outstanding. The nuts added such a nice crunch to this stuffing, and just the taste of the stuffing developed over the roasting time in the oven. I thought we might have extra, but DS came over and well....that was the end of the stuffing. Plate licked clean, and "do we have anymore" was asked. Another purrrfect Chef floWer recipe, thank you!
See Discussion:2 Reviews
Ingredients Nutrition
- 60 g butter
- 1 onion, chopped
- 2 garlic cloves, crushed
- 465 g brown rice, cooked
- 185 g dried apricots, chopped
- 80 g unsalted cashews, chopped
- 15 g fresh parsley, chopped
- 2 tablespoons of fresh mint, chopped
- 1 tablespoon lemon juice
- salt and pepper, to taste
Directions See How It's Made
- Heat 60 g butter in a pan, add 1 chopped onion and 2 crushed garlic cloves and cook until golden. Transfer to a large bowl and allow to cool. Add 2.5 cups (465 g) cooked brown rice, 1 cup (185 g) chopped dried apricots, 1/2 cup (80 g) chopped unsalted cashews, ¼ cup (15 g) chopped fresh parsley, 2 tablespoons chopped fresh mint, 1 tablespoon lemon juice and salt and pepper to taste.
- Stuffing is enough for a 4.5 kg turkey.
- Suitable for roast turkey or duck.

subset: commoncrawl


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Aspen Fall Sports
Click HERE to view the Fall Sports schedule.All fall sports practices will begin on Monday, August 15th, except for Boys Golf which will start on Monday, August 7th. All athletes must have 5 current forms at Aspen High School:(No athlete may participate without all online registration completed)Sports PhysicalsMountain Family Health Center of Glenwood Springs and Basalt are offering $40.00 sports physicals with appointments. Remember a sports physical is good for 365 days only. Sports physicals must be on file before you can participate in practice for any school sport.


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M a r k C o x P a i n t i n g s
C a r m a r t h e n A r t i s t M a r k C o x
Mark Cox is an established artist working and living in West Wales. Mark enjoys painting many and varied subjects in both oils and watercolours but he specialises in painting the landscape and people of Wales. Mark has an honours degree in fine art and values the traditional skills and techniques that can only be learnt with years of study and experience.
"I am a very lucky man being able to work as an artist, something I am passionate about. It is easy to be inspired by the landscape of Wales where there is a new painting around every corner."
Copyright © 2008 Mark Cox. Wales
The contents of this site may not be reproduced in any form without the permission of the Artist


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The spokesman for the president of the United States was unrepentant, saying: "That's our opinion."
Henry turns out to be a complete rat and an unrepentant one at that.
The Obama administration is trying to provide just that, but the efforts of unrepentant hawks are rendering that more difficult.
Opie is devastated, Anthony is unrepentant, and their fans are livid and seeking revenge.
Simon Johnson on why unrepentant former Citi titans Sandy Weill and Robert Rubin also need a day in the court of public opinion.
He is unrepentant enough to believe that he, and not the man with a pencil, is going to "come out on top."
Now that she had time to think over her own tragedy, she was unrepentant.
Burke's voice came with unrepentant noisiness against the stricken man.
But he looked at her, unrepentant, with the light of triumph in his eyes.
Days passed; and he went about his lessons and his work, silent, grim, and unrepentant.
early 13c., from Old French repentant "penitent" (12c.), present participle of repentir (see repent).


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alpine wines
Our wines are an expression of the season. Each vintage may herald slightly different bottlings, and even wines that we tend to make every year will show variation from vintage to vintage. This intrigues us. We work in the field and cellar as minimally as possible, as guides and companions. We believe that terroir encompasses geology, geography, microclimate, varietal, culture, and the human hand. Wine cannot make itself just like a dish of roasted carrots or an aged cow's milk cheese cannot. Our job is to accompany and support the wine throughout its life in the vineyard and our cantina.
Our fruit is handpicked and sorted, foot crushed through pigeage. We employ glass demijohns and old barrels. We hope some day to have an anfora. We rely on the native yeast found on our fruit, the result of a happy marriage of field and fermentation. We use little to no sulphite at bottling. It depends on the wine and the season. Currently in release, we have the first of the 2014 wines with more on the way.
***Please note that we have wines in the marketplace that may no longer be available through the farm. Please see stockists nearest you for inquiries about what might be available to you locally.
We currently do not have a license to ship wine, but you can order wine to be shipped from the stock available at Frankly Wines in NYC. {franklywines.com}
Ci Confonde petillant naturel White 2014 {SOLD OUT}
Ci Confonde petillant naturel Rose 2014 {SOLD OUT}
Grace and Favour petillant naturel white 2014 {London Only}
Damejeanne 2014 {SOLD OUT}
Harlots and Ruffians white field blend 2014 {SOLD OUT}
Loups Garoux 2014 {NOT YET RELEASED}
Lupo in Bocca rose field blend 2013: {SOLD OUT}
Cybele Champagne-method sparkling white 2013 {NOT YET RELEASED}
Vinu Jancu 2014 {AVAILABLE}
Grace & Favour white still 2013 {NOT YET RELEASED}
Bouleverser Cidre Mousseux {NOT YET RELEASED}
If you are interested in our wines, please inquire! Just contact us through e-mail.


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[…] 2. 11 Creative Public Bench Designs – Cool Idea for Public Spaces […]
11 Creative Public Bench Designs – Cool Idea for Public Spaces
How many ways to make a public bench creative and unusual? More than you may have thought. In this post, we will showcase 11 Creative Public Bench Designs collected from the world. Some of these emphasize artistic execution over functional stability. Others take ideas of function to the absolute extreme, which means you have to share your bench with bicycles. From artful to artisan, tentacle to transforming , have a seat and enjoy!
1. Ass Shape Bench [link]
People report that in Kazan there appeared new benches in public places. They look like the one on the photo above.
2. URBAN FURNITURE SYSTEM [link]
This urban furniture system consists of public bench, dust-bin, street lamp, clock, outdoor advertising and urban pylon. The solution presented encourages one to use the dust-bin by combining it with a bench in a hybrid construction, integrating these two functions.
The system is extremaly flexible and versatile by the repetition of the same construction base in all elements, enabling creating any urban furniture combination, e.g. public bench with dust-bin, street lamp with two benches or bench with urban pylon, etc.
3. Two-legged self-supporting Bench [link]
This piece is a parasitic prototype, which cleverly supports itself by gripping into the masonry work of the walls it leans against. Harbert's idea is that with lightweight and mobile furniture, urban landscapes can quickly be transformed into community spaces.
4. Sang-Hoon Lee's Swing Bench [link]
Sang-Hoon Lee's Swing bench brings to mind that episode of FRIENDS, where Rachel is petrified of the swing and how Ross helps her overcome her irrational fears. For the life of me, I can't imagine my childhood without the thrill of swinging in the park with innocent abandonment. Now that I'm all grown up (and a few pounds heavier), the kids won't just let me take the joyride again. I guess I'll have to make do with the Swing Bench to satisfy my urge to be a kid again.
5. The Rolling Bench [link]
The Rolling Bench looks like an ordinary bench with the exception of a winding handle. You can find it on the right side of the bench. The handle rotates the seat surface to expose the dry/clean one. So, after a rain shower, just turn the winding handle and, please, have a sit!
6. Union Bench [link]
This is a bench without a back or front, that let's everyone sit and enjoy from every internal mood and external angle. It's a non- fuss design with maximum practical benefit and desirable, minimalist aesthetic.
7. Wave Bench [link]
project by shigeru uchida. contemporary japanese designer shigeru uchida plays with the 'new wave'.
8. Stone Bench [link]
project by thomas sandell. The art of japanese garden is closely associated with the art of the stone arrangement which are both integral parts of its architecture. giant stones made of corianby dupont by thomas sandell.
9. Piano Park Bench [link]
Designed by the creative minds at ADD Innovation. Piano is a combined bikestand and park bench. The 90 degree angels creates an interesting and effectfull contrast in the landscape.
The bench will come in three different variations.
10. Infinite Bench [link]
project by ron arad. Design is also about the ephemeral nature and transience.
ron arad had the idea of a natural 'loop'.
11. Kajen Public Bench [link]
Designed by Thomas Bernstrand, The Kajen Public Bench is a very interesting & comfortable public seating idea. One of the first park benches to be accompanied by a footstool, the design invites those seated to put their feet up, or just sit back and relax. The laid-back luxury of this bench makes a unique addition to any public space.
6 Comments + Add Comment
im looking for blogs to visit. now i found one, i will surely add it on my rss feeds. definitely i will subscribe on your post.
[…] 8. Sang-Hoon Lee's Swing Bench [link] […]
very nice design go ahead
ha ha thats hilarious
[…] 8. Sang-Hoon Lee's Swing Bench […]
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Maureen Wright
Maureen Wright is employed as a Research Microbiologist in the Formosan Subterranean Termite Research Unit. She earned a B.S. degree in Microbiology from Xavier University of Louisiana, and a Ph.D. in Molecular Biology from the Department of Microbiology at Louisiana State University in Baton Rouge.
Dr. Wright served as a postdoctoral research scientist at the USDA's Southern Regional Research Center (SRRC). She was subsequently hired as a Research Geneticist at the SRRC and was conducting research on gene regulation in fungi when she joined the Formosan Subterranean Termite Research Unit at its inception. Dr. Wright currently develops biological control agents, and has had success developing the genera Paecilomyces and Metarhizium for use against termites.


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Punctuation Worksheets
From early symbol recognition to semicolon usage, our free printable punctuation worksheets cover a wide range of topics to help your child, tween, or teen become a punctuation expert. Each worksheet offers beautiful illustrations and clear instructions to help your child work independently or with an adult. For more general language arts help, check out all of our writing worksheets.
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- As you may have discovered, kids love to correct adults! One fun way to reinforce punctuation is to write a paragraph with periods, commas and questions marks all in the wrong places and ask your child to correct your work.
- Take turns reading aloud with your child, paying attention to stops (periods), pauses (commas), and inflections (question marks). This will help your child pay much closer attention to these symbols in her writing.
- Take a stack of index cards, and on each card draw all the punctuation symbols your child is learning. Then write down the names of those punctuation marks, one per card. Mix the cards up, turn them face down, and play a version of punctuation go fish!
- Your child can put her newfound punctuation skills to the test with these essay writing worksheets.


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Although his wife died years ago, Chance Walker still blames his cousin Jonathan for her untimely death. So when Jonathan returns from England with the widowed Lady Fancy Merrivale, Chance decides to strike back at his cousin by wooing the beautiful Lady. But revenge is bittersweet for Chance as he and Fancy find themselves hopelessly in love--and in desperate danger.
Genre: Fiction
On Sale: May 15th 2001


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NAWBO-IE January 2013 Breakfast MTG
National Association of Women's Business Owners - Inland Empire Chapter
Thursday, January 17, 2013 at 8:30 AM (PST)
Event Details
Communication for Business Owners:
They Don't Hear What I'm Saying
Featuring Timothy P. Beck, Coach, Trainer, and Master Practitioner NLP
Does it sometimes seem like THEY don't understand what you mean? Do you wonder if you're missing the "buy" signs from prospects and clients? Attend the NAWBO-IE January Breakfast meeting and learn how to communicate in a way that gets results--both professionally and personally. January is a perfect time to set your goals for 2013! Join with us and get a jumpstart on these goals by learning some clear communication techniques to communicate with anyone, anywhere, anytime!
Attend and Discover:
- How we communicate with each other and how to use that knowledge to your benefit
- Communicate with anyone, anytime, anywhere
- Pick-up non-verbal cues and know what they mean
- Use more than just your voice to get your message across
- Get a brief glimpse into the world of Neuro-Linguistic Programming
Start the year off right. Attend this great program and networking opportunity! Register now!
NAWBO Inland Empire….
Committed to Adding Value to Your Bottom Line!
Timothy P. Beck
The New YOU Academy, LLC
At the tender age of eleven, Timothy Beck picked up a book entitled "The Structure of Magic." Being a "geek," he thought it was about magic. What he discovered is that it was about using your brain for a change.
This started him on his search for excellence. Over the years, he's read, listened to or seen such great motivational and inspirational speakers as Tony Robbins, Jim Rohn, Earl Nightingale, Zig Ziglar, Wayne Dyer and many, many more.
As the owner of several successful companies, Tim has used the skills of excellence to help himself and others exceed what they thought was possible and achieve greatness. Today, as the Founder of The New You Academy, he holds sixteen certifications in the technologies of Neuro-Linguistic Programming, Time Line Therapy® and Hypnosis, and has been actively teaching, training and working with clients since 2006.
Tim is passionate about passing the skills of excellence on and is pleased to be able to share with you some of the knowledge he's gained over the years. Timothy is a Coach, Trainer, and Master Practitioner NLP Time Line Therapy® Hypnosis.
How to check out:
We use PayPal as our transaction system. However, you DO NOT need a PayPal account to buy a ticket. PayPal accepts all major forms of credit cards and checks (as direct withdrawal/ e-check) to complete the order.
Other Information:
If you're a first time visitor, you can attend our lunch at the member fee. Email our Membership Chair for more details.
If you are a NAWBO affiliate and do not have the discount code to attend the event, email our Affiliate Chair for your organization-unique code.
If you would like time to set-up a booth with information about your business, purchase the business marketplace ticket in addition to your regular ticket.
When & Where
Eagle Glen Golf Club
1800 Eagle Glen Parkway
Thursday, January 17, 2013 at 8:30 AM (PST)
Add to my calendar
National Association of Women's Business Owners - Inland Empire Chapter
The National Association of Women Business Owners (NAWBO) is the only dues-based national organization representing the interests of all women entrepreneurs in all industries. NAWBO's strength comes from its diverse membership - businesses of all sizes and in all industries, and in all regions of the country.


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Pope Benedict XVI's choice as the new Vatican overseer for religious orders, 63-year-old Brazilian Archbishop João Bráz de Aviz, is seen as a personable if little-known figure with a reputation as a moderate-to-conservative, who apparently comes to his new job without a strong personal agenda regarding religious life.
If so, that alone would make Bráz de Aviz a contrast to the man he replaces, 76-year-old Slovenian Cardinal Franc Rodé, a Lazarist who spent much of his five-year tenure as Prefect of the Vatican's Congregation for Institutes of Consecrated Life and Societies of Apostolic Life battling what he described as a "crisis" in religious life following the Second Vatican Council (1962-65).
Among other things, under Rodé's leadership, the office -- popularly known as the "Congregation for Religious" -- launched an on-going, and controversial, Apostolic Visitation of women religious in the United States.
Bráz de Aviz is not a member of a religious order and sources in Brazil say he's not written or spoken widely about issues in religious life. He's seen as closer to some of the new lay movements in the church, especially the Focolare, an Italian group emphasizing ecumenical and inter-religious unity.
Most Vatican-watchers believe Bráz de Aviz was picked largely on the strength of his nationality and general reputation for reliability, as opposed to any specific interest in religious life. With the recent retirement of Cardinal Cláudio Hummes as Prefect of the Congregation for Clergy, there was no Brazilian in any senior Vatican post, despite the fact that Brazil is by population the largest Catholic country in the world.
A dogmatic theologian who studied in Rome at both the Gregorian and Lateran universities, church-watchers in Brazil have long regarded Bráz de Aviz as a rising star.
For more on Bráz de Aviz, see John Allen's interview: New Vatican official: Want to 'learn from, walk with' U.S. women religious
First named an auxiliary bishop of Vitória in 1994, at the age of 47, Bráz de Aviz was tapped as bishop of Ponta Grossa in 1998, archbishop of Maringá in 2002, and finally archbishop of Brasilia, the national capital, just two years later in 2004. He was widely expected to be named a cardinal in the recent consistory, and his appointment to the new Vatican post makes the red hat virtually automatic the next time around.
Despite that insider appeal, Bráz de Aviz has maintained a low public profile, even in his home country. Yesterday the Brazilian newspaper O Estado de S.Paulo described him as "discrete and not well-known" and the Brazilian media generally buried news of his appointment on the inside pages.
Church sources in Brazil generally describe Bráz de Aviz as a centrist, not part of the progressive liberation theology current in Brazilian Catholicism, but also distant from the church's more traditionalist wing.
One Brazilian journalist who covers the church told NCR that during his time in Brasilia, Bráz de Aviz was seen by supporters of the older Latin Mass as an "enemy."
Despite Benedict's 2007 motu proprio authorizing wider celebration of the old Mass, the journalist said, Bráz de Aviz did not grant permission "easily," and in general "the traditionalists didn't seem to have an easy life while he was in charge."
On the other hand, Bráz de Aviz has also kept his distance from the progressives.
In December 2008, he reportedly threatened to boycott a gathering of Franciscans if Leonardo Boff -- a renowned liberation theologian who left the Franciscan order and the priesthood in 1992 after running afoul of the Vatican -- was on the same program. Church sources said Bráz de Aviz's problem wasn't so much Boff's defense of the poor, but rather his advocacy of a "church from below" in contrast to the hierarchy.
In Focolare circles, Bráz de Aviz is seen as a friend. He's celebrated Masses in honor of Chiara Lubich -- the Italian founder of Focolare who died in 2008 -- and in 2007 he opened a beatification process for Ginetta Calliari, a close friend of Lubich who helped bring the Focolare movement to Brazil in the 1960s.
"Focolare", which in Italian means "hearth" or "fireside," was created by Lubich after the Second World War to promote unity and universal brotherhood. It currently operates in 182 nations with some 100,000 members; in the United States they're known for dialogue with the American Society of Muslims, founded by the late Warith Deen Mohammed.
Bráz de Aviz's ties to the Focolare may help him settle in Rome, since many focolarini, as its members are known, serve in Vatican offices.
Given his background, most observers expect Bráz de Aviz to rely in the early stages on the congregation's number two official, American Archbishop Joseph Tobin, a former superior of the Redemptorist order. If so, that may be good news for American sisters alarmed about the possible fallout of the Vatican investigation, since Tobin has repeatedly expressed support for women religious in the States.
Bráz de Aviz becomes the fourth Brazilian to head a Vatican department, after the late Cardinal Angelo Rossi, who led the Congregation for the Evangelization of Peoples from 1970 to 1984 and later the Apostolic Patrimony of the Holy See, and who at one stage was the dean of the College of Cardinals; the late Cardinal Lucas Moreira Neves, who headed the Congregation for Bishops from 1998 to 2000; and Hummes.
If things hold to form, Bráz de Aviz could eventually emerge as a papal contender. Each of his Brazilian predecessors as a Vatican official was widely touted as a papabile in his day, though none, of course, were ever elected.
[John L. Allen, Jr. is NCR Senior Correspondent.]
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IMESD offers hearing screenings to schools
The InterMountain Education Service District (IMESD) is providing free hearing screenings to public elementary school students through November.
Hearing screenings are conducted annually by the IMESD's Audiology Department for public school students in Umatilla, Morrow and Union counties, and is cooperatively sponsored by local school districts. Students to be screened include nearly 6,000 kindergarten, first and third grade students, new students to a county, and parent/teacher/principal referrals of students in other grades. Children who fail the initial screening will be re-screened within a three-week period.
Screenings began earlier this month.
Parents who do not want their child's hearing to be screened should notify their child's school as soon as possible. If you have any questions regarding the hearing screening program, please contact your local school principal or the IMESD Audiology Department at 541-966-3140.
Please click here for the hearing screening schedule.
Accessibility Statement - The InterMountain ESD is committed to making its electronic and information technologies accessible to individuals with disabilities. We are currently in the process of upgrading our web site to ensure we meet or exceed the World Wide Web Consortium's (W3C's) Web Content Accessibility Guidelines (WCAG) 2.0 Level AA and the Web Accessibility Initiative Accessible Rich Internet Applications Suite (WAI-ARIA) 1.0 for web content.
For more information on how to report an issue related to the accessibility or how to file a formal Section 504 or ADA (Title II) complaint about the accessibility of any content on an InterMountain ESD website, please see our full Accessibility Statement.
Anti-Discrimination Policy - It is a policy of the InterMountain ESD that there will be no discrimination or harassment on the grounds of race, color, sex, marital status, religion, national origin, age, sexual orientation, or disability in any educational programs, activities or employment. For more information, visit the Anti-Discrimination Policy page.


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News By Tag
Industry News
News By Place
Industry News
Join the Welsh Male Voice Choir for an afternoon of song at Logwood Village
Enjoy a musical afternoon with amongst others, the Welsh Male Voice Choir. Sing Christmas carols led by St Francis' church choir. This is a fundraiser for Logwood, a home for intellectually disabled people. Bring your picnic baskets and blankets.
A number of well-known musicians are getting together on the first Sunday in December to get you into the Christmas spirit while performing old favourites in a country setting - and all in the aid of a good cause.
The venue for what is billed as a picnic afternoon is Logwood Village, a home for intellectually disabled adults from all over Gauteng. Residents of the home are throwing open their spacious grounds, with idyllic views over the Magaliesburg, for a Christmas concert with a difference.
On the bill are the world-famous Welsh Male Voice Choir, Paul Obermeyer and Rianda. They will be joined by Parktown Girls High, who has had Logwood as one of their charities of choice during the year. And to end the afternoon you will have the opportunity to join in community carol singing with Ruth Coggin and members of Parkview's St Francis church choir and Logwood's own choir.
The event will take place from 15h00 on 2 December 2012 at Logwood's premises situated at Plot 100, Sunset Drive, Nooitgedacht, Muldersdrift. GPS Coordinates:
"Bring your picnic baskets and blankets and join us for a relaxing afternoon in the countryside,"
"It's going to be loads of fun for the whole family and I'm sure it's going to put us all in a really festive mood.
"Should you still be hungry or thirsty after munching on the treats you have packed in your picnic basket, we will be selling Ice cream and cold drink and giving out popcorn. Tea and coffee will also be available".
Says Sprong: "Please help us raise funds for our amazing residents who, despite their disabilities, embrace life to the full."
For more information, contact Logwood on 011 659 0480/0510/0586 or contact Ronell Sprong on 082 414 3260 or Lorraine on 076 970 3669.
The event is aimed at raising funds for the home which provides holistic care for its residents in a sustainable, protective environment.
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This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
Molecular Imprinting Technology (MIT) is a technique to design artificial receptors with a predetermined selectivity and specificity for a given analyte, which can be used as ideal materials in various application fields. Molecularly Imprinted Polymers (MIPs), the polymeric matrices obtained using the imprinting technology, are robust molecular recognition elements able to mimic natural recognition entities, such as antibodies and biological receptors, useful to separate and analyze complicated samples such as biological fluids and environmental samples. The scope of this review is to provide a general overview on MIPs field discussing first general aspects in MIP preparation and then dealing with various application aspects. This review aims to outline the molecularly imprinted process and present a summary of principal application fields of molecularly imprinted polymers, focusing on chemical sensing, separation science, drug delivery and catalysis. Some significant aspects about preparation and application of the molecular imprinting polymers with examples taken from the recent literature will be discussed. Theoretical and experimental parameters for MIPs design in terms of the interaction between template and polymer functionalities will be considered and synthesis methods for the improvement of MIP recognition properties will also be presented.
Molecular Imprinting Technology (MIT) is today a viable synthetic approach to design robust molecular recognition materials able to mimic natural recognition entities, such as antibodies and biological receptors [
The design of synthetic materials, which are able to mimic the recognition processes found in nature, has become an important and active area of research making in recent years molecular imprinting one of the strategies followed to create materials with recognition ability comparable to the natural systems.
MIT is considered a versatile and promising technique which is able to recognize both biological and chemical molecules including amino acids and proteins [
The main advantages of molecularly imprinted polymers (MIPs) are their high selectivity and affinity for the target molecule used in the imprinting procedure. Imprinted polymers compared to biological systems such as proteins and nucleic acids, have higher physical robustness, strength, resistance to elevated temperature and pressure and inertness towards acids, bases, metal ions and organic solvents. In addition, they are also less expensive to be synthesized and the storage life of the polymers can be very high, keeping their recognition capacity also for several years at room temperature.
The scope of this review is to provide a general overview on MIPs field, some significant aspects about preparation and application of the molecular imprinting polymers with examples taken from the recent literature will be outlined. The discussion will evaluate some theoretical and experimental parameters for MIPs design in terms of the interaction between template and polymer functionalities, and synthesis methods for the improvement of MIP recognition properties. In the present review various MIPs applications will also be considered focusing on more recent separation techniques, sensors and biosensors, catalysis and drug delivery.
As reported above, molecular imprinting is a technique to synthesize highly cross-linked polymers capable of selective molecular recognition. The principle is summarized in
For their insoluble nature, MIPs are notoriously difficult to characterize. However, some analytical techniques can be utilized for their chemical and morphological characterization, including solid-state NMR techniques [
Morphological characteristics of MIPs can be properly investigated by microscopy techniques, such as light microscopy to verify the natural integrity of polymer beads and scanning electron microscopy (SEM) to image polymer macro-pores [
However, a very important level of characterization for MIPs concerns the molecular recognition behavior, such as the binding capacity, and one of the best methods for evaluating binding capacity and selectivity is the batch rebinding. MIPs can also be used directly as chromatographic stationary phases which generally provide a quicker and easier method for analyzing the binding properties of MIPs [
An even better MIP evaluation than imprinting factor seems to be the selectivity for a different molecule, but structurally related to the template, because often the presence of the template during polymerization synthesis causes general changes in the morphology of the polymer, such as porosity and surface area, which are confirmed from binding differences compared with the polymer control that may not be exclusively due to the presence of imprinted sites. On the other hand, the imprinting factor must be considered along with selectivity results since a better or less good retention of a certain compound over another one may just be due to their particular physicochemical properties rather than to specific imprinted sites.
Good interactions between monomer and template represent a preliminary essential condition to obtain MIP networks with potential recognition sites. Today there are two main strategies employed for MIP technology depending on the nature of prepolymerization interactions between template and monomer:
self-assembling approach [
preorganized approach [
Amongst these, self-assembling is still the most frequently adopted approach for the preparation of MIPs due to the simplicity of complex formation and dissociation and the flexibility in terms of available functional monomers that can interact with almost any kind of templates. On the other side, the related MIPs have lower binding affinity than those prepared using covalent methods. Hwang and Lee [
Various works reported in the literature demonstrated that prepolymerization studies on self-assembling systems can be useful for the selection of suitable functional monomers and solvents for specific template molecules. The formation of functional monomer-template complexes has been investigated by spectroscopic and theoretical approaches, that includes UV-Vis and NMR spectroscopies and theoretical models as well [
Job's method, titration curves and binding isotherms are currently used to determine the nature of the interactions, the coordination number of the complex monomer-template and the association constant by using spectroscopy techniques. For example, the formation of the complex between nitrofurantoin with 2,6-bis(methaacrylamido) pyridine (BMP) was studied by NMR spectroscopy revealing a hydrogen bonding interaction between the imide moiety of nitrofurantoin with the protons of the pyridine moiety of BMP and allowing also the calculation of the association constant. Similar studies were also reported between carboxyphenyl aminohydantoin (CPAH) and 2,6-bis(methacrylamido) pyridine (BMP). Titration curves were performed and the binding constant was calculated using nonlinear curve fitting for a 1:1 complexation model and the complex stoichiometry was independently confirmed by a Job plot method [
In 2009 Del Sole
In another work [
Pietrzyk [
Finally, in recent works it was demonstrated that all components (template, functional monomer, solvent, initiator, cross-linker) in a prepolymerization mixture can affect template complexation [
In the synthesis of MIPs, many parameters have to be assessed since they can influence morphology, properties and performance of the polymers. Even if many authors have tried to investigate and understand the role of different parameters in MIPs preparation, a rational comprehension of all of them is still quite difficult to achieve and represents a critical point in MIP field; however, some remarks in MIPs synthesis can be highlighted [
Today, the most common method used to obtain MIPs is the free radical polymerization. Generally, the synthesis procedure is performed under mild reaction conditions (e.g., temperature lower than 80 °C and atmospheric pressure) in bulk or in solution, and it is tolerant for a wide range of functional groups and template structures. The polymerization reaction is normally very rapid; it is started by an azo-initiator, commonly azo
In the synthesis of MIPs, the choice of the chemical reagents is of primary importance in order to obtain efficient functional MIPs. A wide range of template molecules such as drugs, amino acids, carbohydrates, proteins, nucleotide bases, hormones, pesticides and co-enzymes have been successfully used [
The choice of monomer is very important in order to create highly specific cavities designed for the template molecule. Typical functional monomers (
In imprinted polymers synthesis, the cross-linker also fulfils important functions. The cross-linker is important in controlling the morphology of the polymer matrix, serves to stabilize the imprinted binding sites and imparts mechanical stability to the polymer matrix in order to retain its molecular recognition capability [
Moreover, nature and volume of the solvent play also an important role in the molecular imprinting process. The most common solvents used for MIPs synthesis are toluene, chloroform, dichlorometane or acetonitrile. The solvent serves to bring all the components (monomer, template, initiator, cross-linker) into one phase in the polymerization and it is responsible for creating the pores in macroporous polymers. The solvent should produce large pores to assure good flow through properties of the resultant MIP and increasing the volume of the solvents, the pore volume of the polymer enlarges. For this reason it is quite common to refer to the solvent as the "porogen". The porogen in a non-covalent imprinting polymerization should also be chosen considering its role in promoting template-functional monomer complex formation: less polar solvents such as chloroform or toluene increase the complex formation facilitating polar non-covalent interactions such as hydrogen bonds; more polar solvents tend to dissociate the non-covalent interactions in the prepolymer complex, especially protic solvents that afford a high degree of disruption of the hydrogen bonds. However in some papers efficient MIPs have been prepared in rather polar solvents (e.g., acetonitrile/water or methanol/water) since strong template-monomer interactions have been observed. For instance, a water-compatible imprinted polymer with strong affinity for a polar 1-methyladenosine template has been prepared in acetonitrile/water 4/1 and successfully used as solid phase extraction (SPE) sorbent for the analyte extraction from spiked human urine samples [
Usually apolar, non-protic solvents, such as toluene or chloroform, are preferred in MIPs synthesis whereas, if hydrophobic forces are involved in the complexation process, water or other protic solvents could be selected as well. Moreover, it was observed that, after the polymerization, the rebinding performance is optimized when carried out in the same solvent used for the imprinting, suggesting that the optimum rebinding conditions require the same or very similar solvation conditions used for the polymerization.
MIPs can be prepared in a variety of physical forms, using different method, depending on their final application [
Regular beads can instead be obtained using precipitation polymerization method. This technique allows the formation of imprinted beads with the same reaction mixture used in the bulk method except for the presence of a higher amount of porogen. Polymer chains will continue to grow, precipitating only when become large enough to be insoluble in the reaction mixture. Then the polymer beads are easily recovered by washing and centrifugation operations. This technique is easy, less time consuming than bulk polymerization and provides regular beads in good yields. The particles diameters decrease, increasing the porogen volume probably due to the formation of less oligomers and nuclei under diluted conditions and with an increase of flow ability of the polymer, thus less monomers and cross-linkers diffuse to the surface and polymerize into the small particles. In a recent work [
In recent years molecularly imprinted polymer membranes have attracted significant interest. Porous free-standing molecularly imprinted polymer membranes were synthesized by the method of
Monolithic MIPs have also been prepared by a simple, one-step,
Surface grafting of MIP layers onto preformed beads has also been proposed as a technique to obtain chromatography-grade imprinted polymers. In this method, thin imprinted layers have been used as coatings on chromatography-grade porous silica or spherical polymers using several techniques to retain the radical polymerization at the surface of the beads.
Molecularly imprinted monolayers have also been prepared for sensor applications. Lahav
Molecular imprinting process presents various advantages, as described above, but some drawbacks must also be considered.
Design of a new MIP system suitable for a specific template molecule often requires a lot of time and work for synthesis, washing and testing. Generally many attempts need to be made, changing various experimental parameters, before finding the optimum conditions. Combinatorial chemistry has been recently adopted in order to accelerate the optimization of MIPs to attain the desired performance [
Another important critical point is the design of water-compatible MIPs. For various applications especially in clinical and environmental fields (for instance many biomolecules are insoluble or lose their activities in organic solvents) the imprinting capability of MIPs in water solutions is required. Unfortunately water molecules will compete with the template making weaker or destroying non-covalent interactions (electrostatic, hydrogen and van der Waals bonds) between template and functional monomer. In spite of this, some progress has been made in the development of water-compatible MIPs. Hydrophobic, ionic or metal co-ordination interactions are proving to be very promising to enhance template and functional monomer association in water. Polymerizable-cyclodestrines were used as the hydrophobic part able to control electrostatic interactions between template and monomer from water interferences [
The peculiar properties of MIPs have made them a highly interesting tool for different application areas, including separation sciences and purification, sensors and biosensors, catalysis and drug delivery.
Molecular imprinting chromatography is one of the most extensively studied application areas of MIPs, which are highly suitable for chromatographic separation, allowing the preparation of tailor-made supports with predetermined selectivity. The increasing demand for optically pure compounds makes MIPs for chiral separations another important application especially to obtain the racemic resolution of drugs [
Recently, several studies have demonstrated that MIPs can serve also as artificial binding mimics of natural antibodies and can be used as recognition elements in immunoassay-type analyses. To date MIPs have been successfully used with different types of transducers and several methods have been used to achieve a close integration of the transduction platform with the polymer [
Considerable investigations have also been made on the use of MIPs as active materials for catalyzing some reactions. MIPs with catalytic properties can be considered mimics of natural enzymes and applied in enzyme-like catalysis [
Finally, an area which poses the greatest challenge for MIPs is that of therapeutics and medical therapy. The capability of the MIPs to bind bioactive molecules in specific conditions makes MIT a huge potential for creating suitable dosage forms.
In the next four paragraphs, present and future prospectives of the most important applications will be discussed.
Molecularly Imprinted Chromatography is one of the most traditional applications of molecularly imprinted polymers [
MIPs were frequently used as Chiral Stationary Phases (MIP-CSPs) in High Performance Liquid Chromatography (HPLC) to obtain enantiomeric resolutions of racemic solutions of different compounds [
It is worth noting that one of the main drawbacks observed in MIPs for HPLC columns, that limit its commercial application, is the excessive peak broadening and tailing. In fact, they are often found in chromatograms for templates when MIP is used as sorbent and they are attributed to the heterogeneity of the binding sites in the MIPs [
MIPs have also been used as media for Capillary Electrochromatography (CEC). CEC is a hybrid separation technique that combines the stationary phase of LC with the electrosmotically driven mobile-phase transport of electrophoresis. MIP-based micro-columns for CEC are recently realized for separation of several compounds [
Solid Phase Extraction (SPE) is another important area of application of MIPs in analytical chemistry [
The principle of MISPE is based on the same main four steps as classical SPE: conditioning of the sorbent, loading of the sample, washing away interferences and elution of the target analytes (
MISPE was applied for extraction of several compounds in different sample matrices such as biological [
The first application of MISPE was made by Sellergren in 1994 which prepared a selective extraction of pentamidine, a drug used for the treatment of AIDS-related disorders, in urine samples [
Recently, some MISPE for vitamins and nucleoside, derivatives from biological matrices, were prepared. A highly selective MIP for 1-methyladenosine (1-MA), an urinary modified nucleoside which is used as cancer marker, has been prepared and used as sorbent material in an off-line SPE mode [
In another study [
Javanbakht and co-workers have recently studied the extraction of important drugs from biological samples, such as plasma and urine. They followed a new water-compatible methodology for determination of the analgesic Tramadol from biological fluids, using MISPE as the sample clean-up technique combined with HPLC [
MISPE procedure combined with HPLC was also applied to extract Testosterone and Progesterone and other important drugs from urine [
In recent years, MISPE was extensively applied also for the determination of analytes in environmental samples. In particular, phenolic compounds were widely studied for their pronounced toxicity and their wide distribution in several environmental samples. In the last few years, a new class-selective sorbent for SPE to pre-concentrate the environmental pollutants, phenolic compounds, from spiked water samples was developed by Peipei
On-line MISPE coupled to reversed-phase HPLC was also applied to selective extraction of 4-nitrophenol in real water samples [
It is well known that template residues are present in the polymer matrix even after exhaustive washing steps and leakage of the template can affect the accuracy of detection in trace analysis. To overcome this bleeding problem the MIP can be synthesized by using a close structural analogue of the analyte as template (also called dummy template) that can capture also the target analyte since very similar cavities are generated [
Several studies applying imprinting technology to different selective extraction techniques have appeared over the last few years such as Solid-Phase Microextraction (SPME), Stir-Bar Sorptive Extraction (SBSE) and Matrix Solid Phase Dispersion (MSPD) techniques.
The first work on SPME appeared in 1990 [
MIPs have been used as coating agents also for Stir-Bar Sorptive Extraction (SBSE) technique [
Matrix Solid-Phase Dispersion (MSPD), commonly used for solid samples, is another extraction technique which differs from the previous ones mainly since the extraction can also be performed directly on solid, semisolid and viscous samples. In this technique the sample of interest and the sorbent are blended together; then, the obtained mixture is packed into an empty column or a SPE cartridge or a syringe. Finally, the analytes can be extracted through two different ways depending on their affinity towards the sorbent used. When there is a high retention of the analyte of interest on the sorbent, a solvent can be added into the cartridge to remove the interfering compounds; while if the analyte of interest is not strongly retained on the sorbent, a solvent able to disrupt only the weaker interactions can be added to directly remove the analyte. In MSPD extraction method, any commercially available sorbent can be used. Some authors have also tried to combine the advantages that MIPs offer for selective extraction with the simplicity of the present technique [
In the last years, chemical sensors and biosensors are of increasing interest in the field of modern analytical chemistry, as can be seen from the growing number of published papers. This is due to new demands that are appearing particularly in clinical diagnostics, environmental analysis and also in food analysis. Recently, a big effort to synthesize artificial receptors capable of binding a target analyte with comparable affinity and selectivity to natural antibodies or enzymes has been done.
MIP technology can also be used as antibody-like materials with high selectivity and sensitivity, owing to their long-therm stability, chemical inertness and insolubility in water and most organic solvents [
Moreover, polymers can also be electropolymerized on the surface of transduction platform. In this case, there are some adhesion problems, especially during the washing process of the polymerized MIP-coated QCM (Quartz Cristal Microbalance) with organic solvents that, sometimes lead to a partly peeling off of the MIP layers produced. For this reason, specific pretreatment to enhance the adhesion of MIPs on transducer platforms, must be done.
The first generation of MIP sensors was prepared using imprinted polymers synthesized in the form of monoliths. The obtained MIP particles were deposited in close proximity to the electrode by incorporation of the particles into the carbon paste of screen-printed electrode [
Competitive amperometric morphine sensor using MIP particulates immobilised with agar gel on the electrode surface, has been reported by Mosbach's group [
MIP-based recognition elements were also prepared as layers or thin films by deposition or grafting onto the transducer platform [
Today, a simple approach to obtain sensor device, is the combination of MIPs with a piezoelectric transducer (e.g., quartz) to create an acoustic sensor called QCM-MIP sensor. The coating of the crystal with the MIP can be obtained by
A QCM-MIP sensor has been employed by Peng and co-workers to obtain a sensor device for atropine detection. In this case, the electropolymerization approach was used to prepare an aniline-co-
Moreover, a sorbitol-based MIP was also prepared by electropolymerization of o-phenylenediamine on a QCM surface [
In another study, an imprinted polymer-coated sensor was created by Tan
Recently, Ersőz and co-workers have developed a new QCM sensor for specific determination of 8-hydroxy-2′-deoxyguanosine (8-OHdG), an oxidative stress marker [
MIPs were also widely employed as sensors for enantiomeric separation of different compounds such as R and S-propranolol,
However, in terms of sensibility, MIP-based biomimetic sensors are still rather inferior to biosensors because they require further optimization of the MIPs and the transducers. An interesting area of research was the preparation of mass sensitive MIP sensors for cells and viruses. In particular, Dickert and co-workers realized a QCM-MIP sensor able to bind with excellent specificity the yeast
During the last decade, the MIP technology has been extensively studied for its potential applications in pseudo-immunoassay [
The competitive formats of MIAs can be divided into two categories: homologous and non-homologous assays, in which analyte and probe are chemically identical or chemically different respectively. Radio-labeled MIAs were the first examples of homologous assays where the probe is just the radio-labeled form of the analyte used as template in the imprinting process. In particular, novel imprinting processes to create spherical, molecular imprinted beads were employed in radio-labeled MIAs as alternatives to conventional MIP particles preparation with several advantages such as enhanced selectivity, low cross-reactivity and a simple bead preparation.
Recently, Ye
Nevertheless, the practical applications of this technology are heavily restrained due to the inherent complications of handling and disposing of radioactive materials. For this reason, fluorescent analogues of analytes were employed as alternative probes in MIAs. Conventional fluorescent-labeled MIAs is an example of non-homologous assay where the MIP is prepared using the analyte itself as template in the imprinting process and a fluorescent probe in place of the radioactive probe. However, a problem that hinders the development of this technique is due to different structures of the fluorescent probe, which has additional fluorescent groups to that of the analyte, inducing a decrease of sensitivity and selectivity. Nevertheless, in the last few years, many researchers have focused their interest on fluorescence-labeled MIAs [
Considerable efforts have been made to investigate the possible use of MIP for catalytic applications [
The use of MIP for catalytic application is very important because MIP catalysts are able to mimic the selectivity and sterospecificity of the binding domains of antibodies and enzymes which are generally utilized as catalysts in several reactions. Catalytically active imprinted materials can be obtained using analogues of substrates, transition states or products as templates in the imprinting protocol [
The strategy using substrate analogues as template, involves the use of compounds that mimic the reaction complex between the substrate and the matrix. The catalytic groups will be introduced in the right positions into the cavities of the polymer and subsequently they will act catalytically in presence of the true substrate. Leonhardt and Mosbach applied this strategy to obtain an imprinted matrix with esterolytic activity using Cobalt (II) ions to coordinate vinylimidazole groups and template during polymerization. Subsequent introduction of the substrates, such as p-nitrophenyl esters of methionine or leucine, into the cavities of polymer, leads to an acceleration and substrate-specific hydrolysis of amino acid analogues [
An alternative approach to obtain the same results was the use, in the polymerization process, of a basic Transitional State Analogue (TSA) and MAA respectively as basic template and acidic functional monomers. The use of the MIP catalyst in the reaction process led to an increase in dehydrofluorination rate compared with the uncatalyzed reaction. The TSA approach was followed by Wulff and co-worker to obtain a phosphonate ester imprinted polymer using amidines as monomers. The amidines have the ability to bind carboxylic acids and phosphonate esters with very high affinity, such that the complexes are almost completely in the associated form, even in water. The catalyst was tested on the hydrolysis reaction of analogous carboxylate esters. High catalytic activity was seen, despite the nonactivated nature of the esters. The reaction rate was 100 times higher for the imprinted matrices compared with the uncatalysed solution reaction [
Very recently, Li
Finally, thermodynamic and kinetic studies were also carried out on MIPs catalytic systems with the aim of understanding molecular imprinting and its specificity [
The ability of MIPs to bind strongly and selectively bioactive molecules makes these materials suitable for their potential application in biological field. The high loading capacity and the prolonged release time of the analytes, such as drugs, amounts to MIPs having a huge potential for creating suitable dosage forms [
In the last years, an area of great challenge in MIP technology is that of therapeutic agents, various MIPs have been used as unusual synthetic polymeric carriers to prepare drug delivery systems [
An efficient drug delivery system should ensure that the drug is released at the right site, in the right dose and for the right period of time [
MIPs for drug delivery applications should have specific characteristics: the imprinted cavities should be stable to maintain the conformation in the absence of the template, but also flexible to facilitate the realization of a fast equilibrium between the release and re-uptake of the template in the cavity. To this end, the non-covalent imprinting provides faster equilibrium kinetics than the covalent imprinting approach [
Imprinted-drug delivery systems have not reached clinical applications yet because the adaptability of molecular imprinting technology for drug delivery requires important remarks on safety and toxicological concerns since the polymer devices will go into contact with biological tissues without causing toxic effects [
Recently, molecularly imprinted hydrogels for the recognition of cholesterol have been prepared by molecular design of methacrylate-based structures containing poly(ethylene glycol) in moderately and highly cross-linked networks. Swelling studies have been done to analyze the cross-linked structures of the prepared hydrogels and recognitive studies have been carried out to evaluate the influence of different reaction conditions on the ability of the systems to absorb cholesterol. The imprinted hydrogels have shown an excellent ability to recognize and release the analyte of interest [
In recent years, many researchers have published about the use of MIPs in drug delivery applications [
The first report of imprinted polymers used as sustained release devices has been presented by Norell and co-workers [
Intelligent drug release, refers to the release of a therapeutic agent, may occur as a result of a specific stimuli such as the presence of another molecule. A system with similar characteristics, able to release testosterone at a rate depending on the concentration of hydrocortisone, was described by Sreenivasan
Molecularly imprinted polymers can be used also to bind several substances in the gastrointestinal tract, blocking their absorption in the body, as a complement of the pharmacological therapy. In this contest, MIPs as chemical traps to remove undesirable substances from the body, such as glucose, cholesterol [
This review has attempted to outline various aspects of molecular imprinting technologies. All considered features present strengths and weaknesses and for any specific application some advantages or problems need to be evaluated.
Important progress in the synthesis and application of MIPs has been reported consistently in the literature within recent years. Low cost, excellent stability and continuously advancing performance of MIPs make these polymers the most promising synthetic materials for molecular recognition in different scientific fields.
New procedures of alternative syntheses for bulk polymerization that can improve the properties of MIPs particles, should promote future development of MIPs in different application fields.
MIPs have been widely studied as HPLC stationary phases, especially for chiral separation. However, in this field one aspect that would enhance performance would be to overcome the broadening and tailing of the template peak that is often observed.
More recently, an increasing number of papers on MISPE have been produced. MISPE is a competitive analysis procedure for the stability and low cost preparation of the imprinted materials, compared with traditional solid-phase extraction. Today there are already MISPE cartridges commercially available for selective extraction of several molecules [
In recent years, particular attention has been directed to the development of MIPs as selective material for sensors and biosensors. However, in terms of sensibility, MIP-based biomimetic sensors are still rather inferior to biosensors because they require further optimization of the MIPs and the transducers. Thus, although the stimulating ongoing work in this field, the commercial development of molecular imprinting sensors is still in its infancy.
Catalysis and drug delivery applications have not yet been thoroughly investigated. Applications in these areas are only just beginning to emerge and there are several potential aspects to be explored that could produce progress in the next few years.
The authors thank the University of Salento (Lecce, Italy) for financial support (60%) and Consorzio INCA.
Scheme of molecular imprinting.
Schematic representation of molecular imprinting of tri-
The most stable prepolymerization complex structures for a ratio of 1:2, 1:3 and 1:4 between nicotinamide and methacrylic acid (Adapted from [
Structure of the most common monomers used for molecular imprinting.
Structure of the most common cross-linkers used for molecular imprinting.
The four steps of the MISPE process.
Chromatograms corresponding to (
MIP catalytic systems developed by Beach and Shea (Adapted from [
Recoveries of nicotinamide in spiked pork liver samples using different SPE sorbents (
|SPE sorbent
|Recovery (%)


text:
Phil Panebianco
Forgotten Heroes - Poem by Phil Panebianco
It's a shame that it takes a tragedy for us to remember and recognize our true heroes.
Not an athlete who gets paid millions just to play a game.
Not a singer or actor.
But the forgotten heroes.
The men and women who put their lives on the line, day in and day out, doing what they love.
Risking their health and safety to help others, just to support their families.
Their families, who dread that late night phone call and told their loved ones are not coming home.
They are just everyday people, doing their jobs.
Yet they always go beyond their call of duty.
Their bond of brotherhood should be an example to us all.
So now, more than ever, they are truly,
New Yorks Bravest and New Yorks Finest.
Let's take a moment out of our lives to remember and thank,
our forgotten heroes.
' Thank you to those who gave some, and some who gave all'
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text:
OK, they botched the title. But not the entire film.
"John Carter on Mars" would tell science-fiction fans (such as yours truly, for example) that this is a sci-fi story. The newly shortened "John Carter" title doesn't tell the general public anything and could be one of the reasons it didn't make a bigger splash in the box-office earnings pool.
If you see this show, you're actually going to the great-granddaddy of many classic science-fiction stories, from "Star Wars" to "Avatar." What makes the movie even more enjoyable, at least for me, is knowing it's based on the writings of Edgar Rice Burroughs (who also penned the famed "Tarzan" series) — writings that are about 100 years old. Most of this is based on "A Princess of Mars," part of the "John Carter" series.
The movie is all about Carter (Taylor Kitsch, "X-Men Origins: Wolverine"), who was a captain in the Confederate army during the Civil War. Carter is a fighter who simply refuses to give up. Regardless of who bullies him or captures him (and this happens several times), we see that he simply does not give up or give out. He's determined to find a stash of gold, in fact.
But his plans go awry when he discovers himself in the midst of a barren landscape where he finds that he is unable to walk at first. The change in gravity has its advantages, though: He easily can leap hundreds of feet in the air. He has a semi-comical encounter with a troop of residents led by Tars Tarkas (voice of Willem Dafoe) and also ends up fighting a battle in which he rescues a princess (Lynn Collins).
The look of several sequences is a reflection of the way Carter and his adventures are depicted in the works of the late artist Frank Frazetta. Fans will recognize and welcome these scenes instantly.
Director Andrew Stanton also helmed "Finding Nemo" and "WALL*E," which are also full of action. He's no stranger to animation, so the computer-generated and motion-capture creatures here have as much substance as their live-action peers.
There's so much action, in fact, that some character development and motivation is omitted, particularly with the secondary and tertiary characters. But it's still great good fun, particularly for Burroughs fans or those who enjoy a fast-paced science-fiction yarn.


text:
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|You are in: UK Politics
Tuesday, 23 January, 2001, 20:00 GMT
Mandelson under fire
By BBC political correspondent Nick Jones
Once again, Peter Mandelson is having to explain himself in damaging circumstances.
He has had to admit that he did speak to an immigration minister about an application for a British passport by one of Hinduja brothers - a conversation that until Monday night he said he hadn't remembered.
Government officials told Mr Mandelson he was incorrect in saying he had not been personally involved because in June 1998 he had a two minute conversation with the then immigration minister, Mike O'Brien.
Downing Street issued the correction, a move the Conservatives' spokesman Andrew Lansley thought inadequate.
"We should have had an accurate reply and an accurate account in the first instance rather than having to drag it out of Mr Mandelson and government spokesmen," said Mr Lansley.
"The government guidelines are very clear. Mr Mandelson at the very least may have made an error of judgement in raising it."
Mr Mandelson did what few other ministers or Labour MPs have said they were prepared to do - raise a citizenship application for a businessman.
'Close link'
It is a step the Liberal Democrats' spokesman Norman Baker thought highly irregular in view of Mr Mandelson's responsibility in 1998 for the Dome and the Hinduja brothers' commercial interest in the project.
"It is no business of Mr Mandelson to make phone calls about people who are not constituents to another minister when he has got a clear link with those people, when he is negotiating with them, presumably to get money for a project to be bailed out, namely the Millennium Dome," he said.
They felt Mr Mandelson's original denial was less than frank and by admitting belatedly to a conversation which he hadn't previously recollected he'd landed Labour in another murky business just as the party was recovering from revelations about a mystery Ł2m benefactor.
Labour MP Alice Mahon thought Mr Mandelson had acted unwisely.
"It shows you can't be too careful and it shows in my opinion that his judgement was slightly lacking and you should really leave it to civil servants.
"I think it's ill-advised and ill-judged, yes."
Mandelson defended
Fellow Labour MP Ray Powell was equally forthright: "Mr Mandelson shouldn't have done it. I've never trusted him since he was adopted as a Labour candidate. And you quote me if you like."
Labour MP Harry Cohen defended Mr Mandelson's right to take up citizenship cases - even from people who weren't his constituents - but he still thought Mr Mandelson had only himself to blame for this latest muddle.
"I think maybe he was a bit unwise not to be open about it but I wouldn't object to his right to do it."
Most of the MPs I spoke to thought Mr Mandelson wasn't in quite as much trouble as he was over the home loan that forced his resignation but they agreed he'd again done himself a great deal of damage.
Downing Street has been firmly on the defensive insisting that nothing improper or exceptional had happened. The passport application hadn't been supported or endorsed - but again Mr Mandelson's political reputation is on the line.
See also:
22 Jan 01 | South Asia
Hindujas questioned by detectives
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