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This report combines microfabricated, multi-compartment chambers and protein-micropatterned surfaces into a new platform for the study of localized, sub-cellular signaling on axon guidance. We focus on the specific question of local crosstalk between N-cadherin and fibroblast growth factor receptor (FGFR) signaling. Motor neurons differentiated from embryonic stem cells extend axons from one compartment through a microchannel barrier into a second compartment containing patterns of N-cadherin against a background of laminin. N-cadherin was effective in both guiding and accelerating motor axon outgrowth. Selective inhibition of FGF receptor (FGFR) function in the axon but not the cell body reduced the rate of axon outgrowth while not affecting guidance along N-cadherin, indicating a differential role of crosstalk between these pathways in helping axons navigate the complex extracellular environment. This approach is readily applicable to other systems of guidance cues.
Proper function of the nervous system relies on the formation of precise connections between nerve cells that span complex substrates and long distances. Axon guidance is a key process in establishing this architecture, in which cells must appropriately integrate and respond to multiple signals presented in the extracellular environment 1. These cues come from a wide range of signaling systems, including "canonical" guidance molecules (e.g. Netrins, Slits, Semaphorins, and Ephrins), morphogens (e.g. Hedgehog, BMP, and Wnt families), growth factors (e.g. NGF, BDNF, HGF, and FGFs), and cell adhesion proteins (e.g. N-cadherin, NCAM, L1-CAM). Extensive cross-talk between these pathways has been demonstrated, introducing additional layer of complexity to the mechanisms controlling axonal navigation. Moreover, guidance molecules that are often associated with extracellular matrix components are received and interpreted by nerve cells in a highly localized manner within the growth cones found at the tips of growing axons. Axon guidance is thus an intricate process occurring on a subcellular scale, requiring highly refined experimental techniques to study and manipulate.
Contemporary microfabrication methods have much promise for enhancing the tools that are used to study how localized signaling drives cell function. For example, Campenot chambers, which consist of millimeter-scale barriers that isolate axons and cell bodies into separate compartments, have been widely used to investigate local signaling by restricting the activity of pharmacological agents to distinct parts of the neuron 2. This allows localized study of receptors on the cell surface and limits the effects of detrimental or toxic drugs to the rest of the cell. Microfabricated versions of the Campenot chamber have been more recently developed that provide higher reproducibility and better compatibility with a broad range of nerve cells by eliminating the need for a sealant compound between the chamber and substrate through which neurons must extend their axons 3-6. The classic stripe assay method, in which matrices are patterned with two different guidance molecules to study axonal substrate preference, has similarly been updated by microfabrication. Specifically, the use of microchannel and microcontact printing methods allow patterning of finer features as well as the combination and alignment of more than two molecular cues 7, 8.
In this report, we combine the benefits of the microfabricated chamber with those of the microcontact printing to study local signaling in motor axons guided along multicomponent protein patterns 8. Our chamber system is modified from previously described systems to accommodate aggregate cultures, such as explants or embryoid bodies containing embryonic stem cell-derived neurons, both being more suitable for the study of axon guidance than dissociated cells. As a first demonstration of this system, we investigate guidance of embryonic stem cell-derived motor neuron axons by N-cadherin, a well-known type I cadherin that mediates homophilic, calcium-dependent cell-cell interactions 9 and plays multiple roles in the vertebrate nervous system development, including stimulation of axonal extension 10-14. These actions are mediated in large part through classic cadherin/catenins interactions. Crosstalk of N-cadherin with FGF receptor (FGFR) and downstream PLCγ/DAG lipase/CAM kinase/Ca2+ pathway has been posed as an additional mechanism 15, 16, global inhibition of FGFR reduces neurite outgrowth on N-cadherin, with no effect on axon outgrowth on other proteins such as laminin 17, 18. However, the precise mechanism of this interaction remains unclear. In this study we utilize the new microfabricated system to examine localization of N-cadherin / FGFR interaction in axon guidance.
The technical goal of this study is to build a system that allows neurons to interact with and choose between multiple biomolecular signals while providing pharmacologically isolated access to axons and cell bodies. As illustrated in Figure 1, our implementation consists of an elastomer (polydimethylsiloxane, PDMS) chamber with two open compartments, one for cell bodies and the other for axons, connected by a series of microscale channels that limit diffusion of chemicals between the compartments while allowing and directing axon growth between these structures. For this study, we chose an optimized set of microchannel dimensions of 500 μm in length, 10 μm in width, and 5 μm in height, with a center-to-center spacing of 25 μm, which supported good fluidic isolation between the two compartments while allowing robust axonal outgrowth. On the cell body side, we introduced into the microchannel barrier a 50 μm tall, 50 μm wide overhang (Figure 1B) to hold the explants in close proximity to the barrier. The microchannels and overhang were cast from a multi-height master fabricated out of photoresist using conventional techniques. The resultant structure was cut to create compartments, and then assembled onto planar glass substrates (modified with protein patterns as described below) to produce the chamber device. The cell body and axon chambers were chemically well-isolated from each other, as illustrated in Figure 2A. The axon chamber was loaded with 10 μM Cy5 fluorescent dye (MW 792 Da) in cell culture media while the cell body side contained media alone. The fluorescence image was taken after incubating the chamber at 37°C for 24 hours. Minimal fluorescence was detected in the cell body compartment, demonstrating chamber isolation. Molecular transport simulations, assuming a typical diffusion coefficient of 1 μm2/msec for small molecular agents and the dimensions stated above, indicate that the concentration in the cell body compartment would reach 1.7% of axon compartment concentration after 24 hours. This limited transport could be further compensated by maintaining liquid level of cell body compartment slightly higher than that of axon compartment 5. Simulations indicate that modest rate of media flow (10 μm/s) created by increasing the liquid depth in the cell body side by 250 μm effectively offsets solute transport; resulting in 0.053% increase in the cell body compartment concentration over 24 hours (Figure 2B & C).
Proteins in the axon chamber are patterned onto the bottom surface prior to assembly. Specifically, we use an indirect affinity approach 7, 8 to avoid drying and potential denaturation of the protein during chamber assembly. Microscale patterns of Protein A are microcontact printed onto the coverslip, which is then aligned with the microchannel barrier using a custom-built aligner (Figure 3A). A slight angle (typically 30 degrees) between the stripes and microchannels is adopted to better detect axon guidance. After assembly, fusion proteins containing the active region of the target protein appended with an Fc domain were captured from solution by the patterned Protein A (Figure 3B). Figure 3C illustrates the capture of Ncad-Fc, a fusion of the extracellular domain of N-cadherin with human Fc. The captured protein concentration was approximately 100 molecules / μm2 as reported previously 8, and this capture is specific, as incubation of a Protein A-patterned surface (blocked with BSA) with fibronectin (which lacks an Fc domain) does not reveal similar patterning (Figure 3D). This indirect capture approach is highly flexible, robust, and, as will be described later in this report, extensible to include multiple proteins.
Figure 4 illustrates the use of these chambers with motor neurons. HBG3 ES cells expressing a GFP transgene under the control of motor neuron specific promoter HB9 were differentiated as Embryoid Bodies (EBs) into spinal motor neurons 19. Chambers were prepared by microcontact printing lines of Protein A and backcoating sequentially with polyornithine and laminin, providing a permissive reference substrate; the activity of surface-captured Ncad-Fc or other target protein is evaluated in comparison to this surface. EBs were then loaded into the cell body compartment and manipulated into position under the overhang using a glass pipette. Figure 4A illustrates the position of EBs against the barrier. Over the course of 48 hours, axons extended from these EBs progress through the microchannels and interact with the target proteins. As illustrated in Figure 4B, axons encountering lines of Ncad-Fc (25 μm width, spaced 75 μm apart) reoriented and followed these lines in contrast to the intervening regions of laminin that did not elicit turning response from motor axons. Axon guidance was N-cadherin specific, as axons on surfaces incubated with pre-immune Rabbit IgG antibody were not similarly guided (Figure 4C). To further demonstrate the specificity of N-cadherin guidance we patterned chambers with a repulsive cue, an Fc-tagged version of EphrinB2 (Eph-Fc). Axons on these surfaces avoided the lines of Eph-Fc, growing instead preferentially along the intervening laminin regions (Figure 4D). We thus demonstrated that the developed system can be utilized to study mechanisms underlying both attractive and repulsive axon guidance.
The remainder of this report focuses on the main strength of our system, the ability to localize pharmacological interventions in order to identify the mechanisms of axon growth and guidance on micropatterned surfaces. We first quantify the guidance along N-cadherin stripes illustrated in Figure 2B by measuring the angle formed between the microchannels and axons. Growth parallel to the microchannels and perpendicular to the barrier edge was chosen as a zero degree reference. Figure 5A shows a histogram of the angle formed by individually identifiable axons on surfaces incubated with either Ncad-Fc or an inert antibody, collected from three separate experiments, measured 48h hours after EB plating. For these studies, the Protein A lines were patterned at an angle of 30 degrees, as notated over this graph, and the increased population of axons extending along this direction illustrates guidance by Ncad-Fc. As a further comparison, Figure 5B reports the fraction of axons extending along the Protein A lines (within a ±10 degree window). The fraction of axons within this window is approximately four-fold higher on Ncad-Fc lines than on lines coated with the control antibody of rabbit IgG (P < 0.001 by ANOVA, n = 3, 179, and 184 axons drawn from three separate experiments).
N-cadherin activity on axon outgrowth has been previously shown to depend upon FGFR signaling, but the localization of this crosstalk is not determined. To first test whether axon's guidance by Ncad-Fc is dependent on FGFR signaling, we measured the fraction of axons aligned along the Ncad-Fc lines (within the same window) in the presence of the FGFR inhibitor, PD173074. This inhibitor selectively inhibits FGFR tyrosine kinase activity, blocking autophosphorylation of FGFR1 20, and also prevents signaling through FGFR2-5. When supplemented at 100 nM (a concentration previously demonstrated to disrupt FGFR signaling 17) into either the axon or cell body compartment, PD173074 does not affect the selective growth of axons along Ncad-Fc lines as illustrated by the last two columns of Figure 5B (P = 0.747 by ANOVA, n = 3, 129, and 168 axons drawn from three independent experiments for inhibitor loaded into the axon and cell body compartment, respectively). Thus, guidance selectivity of axons by N-cadherin is not dependent on FGFR signaling.
We next examined the rate of axon outgrowth on Ncad-Fc as a function of FGFR inhibition. Figure 6 compares outgrowth rate averaged over a 12-hour period, starting at 36 hours after seeding of EBs. When added to the cell body compartment, PD173074 did not decrease the rate of axon outgrowth on Ncad-Fc, compared to that with no inhibitor (P = 0.63, n = 86 and 63 axons for uninhibited and cell-body inhibited samples, drawn from three independent experiments). In contrast, FGFR inhibitor added to the axon compartment significantly reduced the rate of Ncad-Fc stimulated axon outgrowth by 40%, from 43.3±15.9 μm/h to 24.1±10.9 μm/h. (P < 0.001, mean ± s.d, n = 94 axons drawn from three experiments for axon-inhibition). Importantly, axon outgrowth rate on laminin (LN) coated stripes was not influenced by the presence of FGFR inhibitor in either compartment and stayed at a basal level in each condition. These results demonstrate that stimulation of axons on N-cadherin by FGF is localized to within the growth cones and axon.
It is increasingly recognized that signaling within neurons must be considered on a localized, subcellular to nanoscale basis. Most prominently, Campenot chambers have been used to demonstrate that growth cone extension, collapse, and turning are modulated locally within this structure and often involve local protein synthesis 21-23. In this report, an improved, microfabricated Campenot chamber is combined with multicomponent, micropatterned surfaces to investigate the role of localized signaling in growth cone guidance of motor axon outgrowth. We demonstrate that different aspects of axon guidance by N-cadherin occur through different local pathways; specifically, enhanced axon growth rate, but not guidance, uses local crosstalk between N-cadherin and FGFR. Inhibition of FGFR in the cell body did not reduce growth rate, further supporting a model that this crosstalk is local; diffusion of signals downstream of FGFR signaling or FGFR/N-cadherin interaction from the cell body to axon does not contribute to accelerated axon outgrowth. While our report demonstrates the advantages of combining micropatterned surfaces and microfluidic chambers to study axon guidance, the system is sufficiently versatile to facilitate more complex studies of growth cone guidance behaviors in response to three or more signals or to gradients of signals in the axon chamber 8, 24. We anticipate that extensions such as this will provide important insights into molecular mechanism controlling growth cone responses to sequences of molecular cues more closely mimicking the complex substrates encountered by growing axons in vivo.
Microfluidic chambers are fabricated using soft lithography technique 25 using a two-layer fabrication process 4. The device consists of two open compartments bridged by multiple microchannels (5 μm depth, 10 μm width, and 500μm length). A silicon wafer is spin-coated with a first layer of photoresist SU 8-2005 (5μm), and exposed through a mask forming the layout of the microchannels. After development, a second layer of SU8-2050 (50 μm) is spin-coated and exposed through a mask containing patterns of both chambers, resulting in a master with positive relief patterns of the microfluidic device. A pre-polymer mixture of Sylgard 184 (Dow Corning) is cast against the master and cured to obtain a negative replica-molded piece. Two culture compartments are then cut into the elastomer, presenting the final chamber structure. The chambers are plasma treated for 2 minutes before assembling with a patterned coverslip to promote fluidic sealing of the final device
Before patterning proteins, coverslips (25-mm, FisherSCI) were cleaned in Linbro 7× detergent (ICN, mixed 1:4 with deionized water) at 80 °C for 45 minutes, rinsed extensively with deionized water, and baked at 450 °C for 6 hours. Protein A is patterned at specific location of each coverslip through microcontact printing. Briefly, PDMS stamps containing designed geometries were coated with 20 μg/ml Protein A in PBS for 1 hour. For visualization purpose, 1/6 of the Protein A was prelabeled with Cy5 (GE Healthcare). The stamp was washed with DI water and dried by blowing nitrogen gas and placed in contact with the substrate for 10 seconds. The coverslip was further plasma treated for 2 minutes with the stamped region covered by a piece of plain PDMS.
The prepared chamber piece was assembled with the coverslips under our 6-axis aligner, to ensure that the exit of the microchannels is close to the protein A patterns. The entire inner surface of the chamber was blocked with 4% BSA for 2 hours. The axon compartment were then were then exposed to either Ncad-Fc (5μg/ml, R&D Systems) or Ephrin-Fc (10 μg/ml) in PBS + 4% bovine serum albumin (BSA, Sigma) for 45 minutes at room temperature, and finally rinsed with PBS. And then, both compartments were sequentially incubated with polyornithine (Sigma) overnight (10 μg/ml for cell body chamber, 2 μg/ml for axon chamber) and laminin (10 μg/ml, Invitrogen) for 1 hour before loading EBs.
Embryoid Body preparations of rostral cervical MNs were produced by addition of Retinoic acid and Hedgehog agonist to embryonic stem cell lines as previously described 19, 26. Briefly, HB9::GFP transgenic mouse-derived (HBG3) ES cells were grown on mouse embryonic fibroblasts in ES cell medium. ES cell colonies were dissociated after 2 days and cultured in DFK5 media. Media was replaced at 2 days and supplemented with all trans retinoic acid (1μM, Sigma) and hedgehog agonist SAG (500 nM, Calbiochem). Medium was replaced on day 5 of differentiation and Embryoid Bodies (EBs) containing MNs were harvested on Day 6-7 for use in the axon guidance studies.
The cultures were observed by fluorescent and bright-field microscopy using a 10X objective on an inverted Olympus IX71 microscope. During imaging, the temperature and CO2 level were maintained at 37 °C and 5% respectively using a live-cell imaging chamber (Pathology Devices, MD). To quantify the axons' growth speed, images were taken at 12 hour interval to identify the extended length of each axon during that period, and to derive the averaged hourly growth rate.
Simulation of diffusive transport in the channels was done by finite element methods, using the COMSOL software package (COMSOL Multiphysics v3.2, Burlington,MA). The simulations presented here use the convection-diffusion model and assume a diffusion coefficient of 1μm2/ms and media viscosity at 37 °C of μ=6.92×10-4 Pa•s. The initial concentration of chemical in the axon compartment was 10 μM, and set to zero in the other compartment. For calculations of flow versus pressure head difference, the resistance of the rectangle microscale channels, which measured 500 μm in length (L), 10 μm in width (w), and 5 μm in height (h), was estimated as R ≈ 12μL/(h3w(1−0.63h/w))27.
This work was supported by Columbia University through the Research Initiatives in Science and Engineering program. The CEPSR cleanroom at Columbia University is also acknowledged for assistance in microfabrication.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Change A Life, Give a Bike 4 Nepal.
Throughout the month of May, Wheels 4 Life will be putting all donations not already allocated to a specific fundraiser, to bikes for the devastated country of Nepal.
Having been desimated by a 7.9 magnitude earthquake, with over 6,600 people confirmed dead and 14,000 casualties, it is hard to even begin to imagine the suffering of the people. This suffering will only continue and for too long. With millions of men, women and children now homeless, the outbreak of diseases, lack of clean water and sanitation and the need for medical attention; we know one thing for sure, Nepal needs bicycles.
$150.00 will buy a bicycle. We have already worked with Nepal in the past and have a project leader on the ground. We can ensure that every bike gets to the right people. Healthcare workers will be able to travel farther thus treating more people. Water can be transported to ...

subset: commoncrawl


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This time is all about them.
It's pay back time. Your fingers and toes do all the work and never get the reward due. Give them glamour!!,, give them shine!!,, give them color!!.
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Waterfront terrace design with ocean view, this great moment to be there, beautiful view and luxury living. terrace design combined with water and garden, you can seating in around bed with canopy, the hotel terrace include with mini bar, you can enjoy and relax with friend and family. let's see how beautiful Terrace Design in Hilton Hotel in Pattaya, Thailand it's with picture below:
For more information visit the tropstudio | via


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Forums > Hair, Makeup & Styling > Drag makeup1 Any tips?!
I am doing a drag makeup this weekend, it is going to be very glam... of course. I have only done this kind of makeup one before, so I would love to hear some tips and advice. I tend to do quite natural makeup and I know this will not be a case... lol
Mar 15 12 08:31 pm Link
Makeup Artist
Posts: 179
Sheffield, England, United Kingdom
when I had a project to do involving drag make up I turned to youtube, these links will give you the idea, also if you are able to watch it there is also Rupauls Drag Race.
http://www.youtube.com/watch?v=D0lwhOEF … -wyJ8HttA=
good luck with your make up
Mar 16 12 07:51 am Link
Makeup Artist
Posts: 487
New York, New York, US
time to break out the silly putty and sand paper!! haha
But seriously you need a heavy full coverage foundation, and a trick to hid the stubble blueness if there is any, use a cheap red lipstick and blend it into the foundation in the beard area.
I did drag way back when, I used about 5 sometimes more different colors of foundation to shape my face. from white to skintone to yellow to dark,dark brown.
for lashes use 2 sets together per eye ..it makes a big difference.
Mar 16 12 03:40 pm Link
Thanks guys! I will let you know how it goes!
Mar 17 12 12:20 pm Link
SianRJJ wrote:
Thanks for those links! they were very helpful!!
Mar 17 12 04:00 pm Link
CMaquillage wrote:
Thanks so much for the tips!!!
Mar 17 12 04:01 pm Link


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Space travel between different galaxies is hypothetically possible, said astrophysicist Geraint Lewis from Australia. Lewis stated that that spaceship can travel faster than light while still following the Albert Einstien's "General Theory of Relativity". This will be made possible by bending the space such that we can travel from one galaxy to other within short span of time giving an appearance that we traveled faster than light.
According to Lewis, to make it possible we need to build a warp drive that will be made of elements with 'negative density energy'. Scientists believe that such elements are present in the universe. However, they haven't located such materials on Earth. To make the warp drive spaceship, first challenge before scientists is to locate and collect elements with negative density energy.
Lewis further added that empty space in the universe has a negative energy density and we can mine it and shape it according to our needs. But what worries Lewis is that even if we could mine such elements to build the warp, still there isn't any guarantee that it will work and the drive will travel faster than light.
But on papers, i.e. theoretically, researchers can bend space on the basis of Einstien's equation and found that we can travel at different speed at different places in the universe.
If reports are to be believed, NASA is already working on such project where researchers are designing a warp drive spaceship. While explaining, a scientist said that the spaceship would produce warp bubble around it with the help of two gigantic rings to travel between galaxies through space. The spaceship will not travel at a speed faster than light but would warp space and will reach its destination before light giving an appearance that the drive traveled at a speed faster than light.
NASA's Dr. Harold White along with Mark Rademaker revealed the design of the future warp drive spaceship. However, it might take another 100 or 1000 more years to make any such functional warp drive. Who knows that fantasy Star Trek might become reality one day.
Moreover, Geraint Lewis completed his graduation in Physics and Astronomy from the University of London in 1990. He further completed his PhD from the University of Cambridge's Institute of Astronomy. Currently, he is working as a post-doctoral researcher.


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Coopers Inn at Killinick sold for near its €575,000 asking price
Popular licensed premises Ye Old Cooper's Inn at Killinick has been sold for close to its asking price of €575,000.
'From our point of view it is a great test of the market to sell a thriving business like this,' said Colum Murphy, the managing director of Kehoe and Associates.
Mr Murphy declined to comment on who had bought the premises from Padraig Doyle, but the new owner is said by local sources to be well known local businessman Eugene Fortune.
The property comprises a non-residential licensed premises standing on a substantial site. This area includes an extensive tarmacadamed carpark to the front with parking for approximately 50 cars, some adjoining outbuildings and a small paddock on the southern side of the property.
Wexford People


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This fantastic vegetarian quesadilla needs some chopped zucchini, chopped red bell pepper, some chopped yellow squash, chopped red onion, chopped mushrooms, a little olive oil, some cooking spray, whole wheat tortillas, as well as some shredded sharp and reduced-fat Cheddar cheese. It will take about fifteen minutes to assemble the necessary ingredients, and it just needs about 15 minutes to cook the quesadillas. With the quesadillas ready in just half an hour, you can certainly make the most of a premium quality quesadilla maker.
Simply add some olive oil to a large nonstick pan and when heated, add to the oil the red pepper, zucchini, onion, yellow squash and mushrooms. Cook the ingredients using medium to medium high heat for around seven minutes or till the veggies are just tender. Extract the vegetables from the pan and set aside.
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As men, it's normal to be concerned about how we feel and look. This includes being confident as well as secure with the size of our penises. Being able to feel adequate regarding the size of our male members is critical- even if we are average sized. Until the previous decade, there was just too little men could do to have a larger dick. Thankfully, this has since changed considerably as men are now open to a broad range of penis enlargement techniques.
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Positive aspects of penis extenders
Penis extenders are gaining increasing popularity in recent years. This is because of the numerous positive aspects associated with its use. For instance, penis extenders are a 100 percent safe if used according to the instructions provided. The device is designed in a manner that allows it to effectively stretch the penis tissues over a specified period of time resulting in permanent penis size gains. This technique isn't anything new though- only that it has been improved technology wise. It has been in use in the past centuries.
Having A Small Penis? Try Out These Sex Positions For Small Penis.
Another positive attribute of penis extenders is that they are hands off. This means that you can wear it under your clothes for several hours and still carry on with your routine. To get the most out of your penis extender, try wearing it under your clothes several hours daily. The more the extender is worn, the faster you will witness penis gains.
The effectiveness of penis extender isn't only dependent on how well you use it. It is important for you to choose the right device. At the end of the day, you get what you actually pay for- typically the same as buying anything else. I would advise you to buy one of the few popular brands to avoid the frustrations associated with encounters with scams. Also, avoid extenders that are unreasonably cheap- when the deal is too good, think thrice. You never know, they could be made of inferior materials and thus, doesn't measure up to the required medical standards.
That's not all
Commitment and effort are two important aspects that must be present. Just using the penis extender isn't enough to obtain desirable results. The number of additional inches an individual can gain is somewhat dependent on the genes he carries. However, there is more. The user must exert enough effort as well as commitment for the extender to deliver optimal benefits.
Further, don't expect to witness additional inches overnight. It's a process that will bear ultimate penis size gain in a couple of months.
Clean Without Her Asking
Women don't like to have sex in haste or when they are busy. In fact it irritates them. You can help her in finishing her household work, which will be give enough time to relax her mind and body. Clean the house and help her in cooking, without her asking for the same. She will be definitely happy with such a move, and will enjoy talking to you while working. This will help her in bringing out all her sexual desires, something that is not able to do when she is busy. You will get numerous opportunities to be close to her and to kiss her.
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