text:
Toyota Tundra 3.5inch Lift Kit 2007 – 2016
Zone Offroad Products 3.5″ Adventure Series UCA Suspension System. This Toyota Tundra 3.5inch Lift Kit is engineered to take advantage of the factory suspension design, offering smooth on road ride quality while increasing wheel travel when you hit the trails. This improves on Zone's standard leveling kit with top mounted strut spacers, heavy duty upper control arms, bump stop extensions, sway bar and differential relocation brackets, rear shackles to level the stance and your shocks of rear shocks. With clearance for up to 35″ tires, this 3.5″ adventure series system is a more affordable option to the full IFS lift systems.
At the core of this system is a pair of adventure series upper control arms (UCA). Our adventure series arms use a repositioned ball joint mount to provide greater range of motion for improved suspension travel. This corrects the ball joint binding issue common on stock control arms leading to premature wear of ball joints. Control arms are constructed from 1/8″ steel plate, boxed and fully welded with CNC machined ball joint and bushing cups. High-quality MOOG ball joints come pre-installed and ready to handle offroad abuse. These ball joints are greaseable for easy servicing as well as far superior corrosion protection when compared to uniball style control arms. At the frame mount, the arms use factory style, durometer-specific rubber bushings pre-installed for quiet maintenance free. The arms come fully assembled, finished in a durable textured black powder coat finish for quick, easy installation right out of the box.
Also included in the 3.5″ adventure series UCA system is a pair of top mounted steel fabricated strut spacers designed to provide the lift while retaining original ride quality. The top mounted design is simple to install and speeding up installation time by not requiring strut disassembly. With the increased ride height the factory bump stops are relocated with the supplied relocation brackets. Similarly, the sway bar is relocated using drop brackets to retain an operating angle similar to stock. Finishing off the front of this 3.5″ adventure series lift kit are differential drop spacers and longer hardware to keep the CV axle angles at a minimum while still allowing the use of the factory front driveline. For 2wd models, these are not used. In the rear, this kit uses a replacement shackle to provide 1″ of lift to level the vehicle stance. Rounding out the rear is your choice of rear hydro or nitro gas shocks.
Note: The Toyota Tundra 3.5inch Lift Kit is available with your choice of shocks. MAKE SURE to select the correct shocks when placing your order.
- Fully Welded, Heavy Duty Boxed Upper Control Arms That Dwarf The Factory Arms
- CNC-Machined UCA Bushing & Ball Joint Cups
- OE Style Rubber Bushings & MOOG Greasable Style Ball Joints For Quiet, Reliable, & Low Maintenance Operation.
- Improved UCA Range Of Motion Compared To Stock Arms
- UCAs Utilize Replaceable Ball Joints and Bushings
- Top Mounted Strut Spacer Retains Factory Ride Quality & Offers Easy Installation
- Differential Relocation to Correct CV Shaft Operating Angle
- Long Lasting Black Powdercoat Finish
- Allows Clearance for 35″ Tires
- Limited Lifetime Warranty


text:
About the Book
This phenomenal collection of poetry contains 100 original creative works, composed by the author. Readers will be pleased with the diverse offering of poetry in various aspects of day to day life. Not to mention some which provoke thought and introspection.
Features & Details
- Category Poetry
6×9 in, 15×23 cm
82 Pages
- Publish Date Jun 07, 2014
- Language English
- Tags Birthday gift, Christmas gift, best gift, gift help, gift ideas, perfect gift, poetry book, poetry books, creative writing, literary, literature, help, gift, writing, poem, poetry
About the Creator
If asked the meaning of the pseudonym 'Qosmic Qadence', the answer would be simple. "I believe that my vision & purpose in life are 'Infinitely vast' and there's definitely 'rhythmic flow' to my method of operation. I was born to bring to the world a sense of comfort that comes by way of knowing, accepting & then walking in your purpose!" Born Muwwakkil S. Abdul-Qawwee, in Washington, D.C. On Feb. 3rd, Muwie or Qūe (as he's affectionately referred to by family and friends) is a poet, author, song writer, outside of the fictitious box thinker & passionate motivational speaker, who lives to inspire life in others. 'Qosmic Qadence' believes that "Creative writing affords the holder of the pen an opportunity to share insightful craftiness soul generated." To date he has penned over 500 poems, quotes, songs & other inspirational content, on the various aspects of life. "Love vs. The World- A Book of Poetry" released in December 2013, is his first published work, with others Coming Soon.
Log in or create an account to post a comment.

subset: commoncrawl


text:
Sudbury Suffolk Retired
- I have used APH Meet & Greet 4 times over the last year and every time the service has been superb all the hastle is taken out of parking and I would recommend without hesitation.
Published on: 01 August 2017
APH has teamed up with a trusted partner to bring you a fantastic new Meet & Greet parking option at Stansted Airport. APH have worked with the owners of I Love Meet & Greet at Gatwick for more than 20 years, so we know that they are as committed to providing a top quality service as we are!
APH Meet & Greet is a drive in and drop off service that takes the hassle out of airport parking. Say goodbye to the transfer bus, as you cruise up to the terminal. Head for the short stay car park where you will be met by a friendly and fully insured chauffeur who will park your car for you. As your car is carefully driven to a safe & secure car park, you are free to head to the check in desk and begin your trip.
Picking up your car on your return is just as easy. Simply make one quick phone call upon clearing customs and your car will be ready and waiting for you in the short stay car park, as soon as you step outside the terminal.
The car park, located within the airport boundary, is a holder of the police approved Park Mark Safer Parking Award and features barrier entry, CCTV and security lighting. It is manned 24 hours a day so you can rest easy and enjoy your trip, knowing that your car is in safe hands.
Important Information
You will need to provide the following information on booking: Inbound & Outbound Flight numbers, Airport Terminals, Car Details and Contact Telephone number (preferably a mobile number). Minimum stays and charges may apply at this car park.
On your return, your car will be delivered back to the orange short stay car park which has a 10 minute free period. It is therefore essential you are ready to collect your car otherwise you will be liable for any excess car park fees.
As you approach the terminal follow signs for short stay car parking and head for the furthest car park, sign posted orange short stay car park'. Take a ticket and follow the road round to the orange bollards, pull in on the left hand side, park and your driver will meet you there. Please make sure you hand your car park ticket to the driver.


text:
117 Lava St Warrnambool, Vic 3280
Personal Loan Calculator
Calculate the repayments on a personal loan.
Note: The results from this calculator should be used as an indication only. Results do not represent either quotes or pre-qualifications for a loan. South West Credit may not provide loans with the details indicated in the calculator. The specific details of your loan will be provided to you in your loan contract. It is advised that you consult your financial adviser before taking out a loan.
© 2014 South West Credit Union Co-operative Limited | Warrnambool | ABN 44 087 651 705 | AFSL / Australian Credit Licence 241 258 | BSB 803 073


text:
Product Spotlight
Featured Research
Quick Stats
You are here
Circle K and XYIENCE Name Winner of its Harley Davidson Sweepstakes
Sweepstakes involved contest and promotions that both companies deemed a "huge success."
LAS VEGAS -- Circle K Great Lakes Division and XYIENCE, maker of Xenergy energy drink, the official drink of the Ultimate Fighting Championship (UFC), announced that Dolly Charley of Akron, Ohio, was the winner of the Harley Davidson sweepstakes.
Circle K and XYIENCE teamed up for the text-message based contest, where one lucky winner had the opportunity to win a Harley-Davidson Sportster 883 Low motorcycle. The promotion, which ran from Sept. 1 through Oct. 31, 2010, took place in Circle K stores in Ohio, Michigan, Pennsylvania, Indiana, Kentucky, Maine, New Hampshire, Vermont and Massachusetts. During that time the c-store chain offered Xenergy Premium on a 2-cans-for-$3 price promotion, but no purchase was necessary to enter the contest, which according to XYIENCE and Circle K "made the sweepstakes a huge success."
"Our customers relate to the Harley-Davidson brand, so the promotion generated a lot of excitement in our stores," Ken Dillon, senior category manager at Circle K said in a Xenergy press release. "It's important to Circle K that we engage our customers in the most authentic manner possible, while at the same time driving sales. Working with XYIENCE, we were able to successfully accomplish those two goals. On behalf of Circle K, I want to congratulate Dolly Charley, and we look forward to future promotions with XYIENCE."
XYIENCE and Circle K presented Charley with her prize at the Circle K location at 935 E. Tallmadge Avenue in Akron. Charley is a longtime Circle K shopper and points to Xenergy Xtreme Cherry DillRush as her energy drink of choice, the release stated.
"I'm a big fan of mixed martial arts, the UFC and Harley-Davidson, so when I saw this promotion at Circle K it caught my attention," Charley said in the release. "I didn't really think much of it until I got the call saying that I won. I was shocked and thrilled; it's an amazing opportunity."


text:
States flock to Beijing's dance
Related topics
It may once have been a slow waltz in the shadows, but diplomacy between China and its neighbours across the Mekong region is moving at a friskier tempo.
Envoys from Thailand, Laos, Cambodia, Burma and even former enemy Vietnam say bilateral ties with their giant neighbour to the north have never been as strong as in recent months.
Observers say the wooing of Beijing is starting to bring rewards but could yet prove divisive.
The new ties are paying off as China defensively re-aligns its strategic partnerships in the wake of the Belgrade embassy bombing, yet at the same time seeks to dilute US and Japanese economic and political influence. Significantly, it also is diluting the power of Southeast Asia.
'From every angle, we see China with new clout and influence one-to-one across the region. It is a player at all levels whereas once it was a cold outsider to be treated with the utmost suspicion,' a European envoy said.
Increased diplomacy is being matched by increased trade, investment and, in some cases, aid flows. Defence ties are also growing, with some analysts believing that China and Japan are competing to forge a future security pact with the region.
Both Burma and Laos are being eyed as trade gateways for land-locked Yunnan province, while new ties with Cambodian Premier Hun Sen stand to weaken his long friendship with Vietnam.
China has been using the friendship to try to scupper a potentially embarrassing trial of Khmer Rouge leaders.
Last week, Thailand played host to an unprecedented delegation of Chinese 'strategic thinkers' headed by Yang Yan Li, counsellor of the Asian Affairs Department, who did little to mask China's desire to get even closer to the region.
'They made clear US and China ties are stagnating,' one senior Thai official involved in the closed-door sessions said. 'They want to pull us even closer. They constantly stress they don't want to be seen as a possible aggressor any more.' President Jiang Zemin is due to visit Bangkok in September.
Thailand has been at the core of the Chinese regional efforts, with Beijing contributing US$1 billion (HK$7.75 billion) to its International Monetary Fund bailout nearly two years ago - an unprecedented act for a communist country. The Chinese Embassy is also funding cultural developments across Chinatown.
Some diplomats believe the Thai relationship hints at the success of the policy in weakening the recently expanded Association of the South East Asian Nations - a union that had been seen increasingly as anti-Chinese.
Four years ago the grouping stood strong in the face of Chinese incursions into Philippines waters on the edge of the disputed Spratly Islands group.
Over the past year, as China returned to intensify building on Mischief Reef, Manila has struggled to raise the same level of concern - even from fellow claimant Vietnam.
Vietnam only restored links with China in 1991 after a freeze following the brief but bloody border war of 1979. Now Vietnamese officials privately describe China as their most important partner militarily, politically and culturally.
The ruling communist parties of both states are actively swapping notes at all levels on how best to stay in power despite reform.
Most Popular
- 1
- 2
- 3
- 4
- 5
- 1
- 2
- 3
- 4
- 5


text:
|Home | About | Journals | Submit | Contact Us | Français
Elevated levels of tissue crosslinking are associated with numerous diseases (cancer stroma, organ fibrosis), and also eliminate the otherwise remarkable clinical successes of tissue-derived scaffolds, instead eliciting a foreign body reaction. Nevertheless, it is not well understood how the initial physical and biochemical properties of cellular microenvironments, stem cell niches, or of 3D tissue scaffolds guide the assembly and remodeling of new extracellular matrix (ECM) that is ultimately sensed by cells. Here, we incorporated FRET-based mechanical strain sensors, either into cell-derived ECM scaffolds or into the fibronectin (Fn) matrix assembled by reseeded fibroblasts, and demonstrated the following. Cell-generated tensile forces change the conformation of Fn in both 3D scaffolds and new matrix over time. The time course by which new matrix fibers are stretched by reseeded cells is accelerated by scaffold crosslinking. Importantly, stretching Fn fibers increases their elastic modulus (rigidity) and alters their biochemical display. Regulated by Fn fiber unfolding, more soluble Fn binds to the native than to the crosslinked scaffolds. Additionally, matrix assembly of fibroblasts is decreased by scaffold crosslinking. Taken together, scaffold crosslinking has a multifactorial impact on the microenvironment that reseeded cells assemble and respond to, with far-reaching implications for tissue engineering and disease physiology.
While the significance of the physical characteristics of the microenvironment in controlling cell function and fate is increasingly recognized,1–5 relatively little is known about the mechanisms by which such physical properties are sensed by cells and how they help to regulate diverse cell functions. For example, it is not well understood how extracellular matrix (ECM) deposited in vitro by different cell types can have differential effects on the phenotype of cells that are seeded into it: matrix produced by tumor-associated fibroblasts promotes tumor cell migration,6 and matrix produced by bone marrow cells prevents the differentiation of mesenchymal progenitor cells.7 Consequently, efforts to recreate microenvironments characteristic of normal and diseased tissue (e.g. fibrotic tissue, cancer stroma, stem cell niches, mesenchyme) in the laboratory, with sufficient fidelity to study diseases and test therapies on the cellular level, has proved a complex undertaking and exposed gaps in our understanding of cell–microenvironment interactions.8–10 Likewise, synthetic strategies for mimicking the natural extracellular environment for applications in tissue engineering and regenerative medicine have yielded few scaffolds that are able to regenerate functional soft tissues successfully. Possibly the most promising tissue scaffolds today are not in fact artificial, but de-cellularized ECM derived from tissue.11 Even de-cellularized rat hearts, reseeded with cells and exposed to simulated pulsatile flow and electrical stimulation, could regain partial pump function after eight days.12 Clinically, tissue-derived ECMs have been successfully used as scaffolds and grafts for the reconstruction of tendons, arteries, and numerous other soft tissues;11 however, the properties that enable these ECMs to successfully guide tissue regeneration are still relatively uncharacterized. It is also poorly understood why chemically crosslinking tissue-derived scaffolds in order to enhance their mechanical stability typically results in chronic inflammation and fibrous encapsulation rather than tissue regeneration.13,14 More generally, enhanced ECM crosslinking via natural processes, while a critical process in wound healing, is also associated with the pathologies of numerous diseases.15–17 For example, lysyl oxidase, an enzyme that crosslinks collagen and elastin, is up-regulated in fibrosis, but down-regulated in malignant cells.18
How does crosslinking affect the ECM microenvironment, and thereby cell and tissue function? Both the biochemical composition and the physical properties of the cellular microenvironment control numerous cellular processes, including cell differentiation,1,2,4,7,19 and tumor cell development and metastasis.3,5,6,20–22 Initial interactions between cells and 2D substrates are dependent, for example, on substrate rigidity, topography and ligand presentation (as reviewed in ref. 23–28). Later events such as stem cell proliferation and differentiation can be influenced by the conformation of surface-adsorbed fibronectin (Fn)29,30 as well as substrate rigidity.4 In all of these experiments, initial surface characteristics were correlated with cell phenotype, yet the molecular mechanisms by which these initial substrate parameters direct long-term cell fate remain unknown. Indeed, it has long been known that a state of dynamic reciprocity exists in which signals from the microenvironment affect cell behavior and cells modify their microenvironment by, for example, ECM deposition and degradation.31 Given that many cell types begin to assemble their own matrix soon after being seeded onto a surface or 3D scaffold, the question arises whether late cell phenotypes are primarily directed by the initial contact or by the evolving properties of the microenvironment. This lack of knowledge is partially due to the fact that tools were not available that enabled the monitoring of changes in the microenvironment's physical properties in the context of a 3D matrix. In 2D culture, traction forces that cells apply to their microenvironment in early phases have been probed using microfabricated substrates32,33 and the rigidity of matrix that surrounds cells cultured on planar surfaces has been measured by indentation measurements using an AFM tip.34,35
By exploiting a fluorescence resonance energy transfer (FRET)-based method that utilizes FRET-labeled Fn as a mechanical strain sensor,36–38 we have previously shown that Fn strain and unfolding in matrix produced de novo by fibroblasts on 2D glass surfaces is not static, but gradually increases as cells apply force and deposit new Fn fibers. Here, we probed for the first time how cells that are reseeded into a cell-derived 3D scaffold remodel the scaffold and, in response to the scaffold cues, gradually build-up their new matrix environment. By incorporating FRET-labeled Fn, either into cell-derived ECM scaffolds or into the Fn matrix assembled by reseeded fibroblasts, we asked here how crosslinking of the scaffolds affected their characteristics and those of newly assembled Fn fibers during cell-mediated remodeling over time. Recent findings have indicated that cell-generated traction forces are sufficiently high to stretch39,40 and unfold fibrillar Fn.37,41,42 Moreover, force-induced unfolding of the secondary structure of intra- and extracellular proteins has been implicated in the conversion of a mechanical stimulus to a change in protein functionality and/or biochemical signaling.43–48 Previously, we have shown in 2D that the amount of Fn stretching and unfolding is up-regulated by the rigidity of a polymeric 2D substrate against which the cells pull.42 However, mounting evidence indicates that observations made in 2D cell culture are not necessarily transferable to 3D.8,9 For example, cells on 2D substrates versus 3D matrices have a different morphology and adhesion structure,49–51 show differences in cell motility,52,53 and favor different non-muscle myosin II isoforms for force application.54 The molecular strain or force-induced elongation of a molecule directly reads-out the extent to which structural, and thus potentially functional, alterations have occurred.47 Yet, to our knowledge, there is no information about how the mechanical strain of newly assembled matrix relates to the rigidity of a provided 3D scaffold.
As cells pull locally on individual ECM Fn fibers, at least two important fiber parameters are altered: fiber rigidity (kPa to MPa)55 and Fn structure.37,41,47 To our knowledge, no work has made a systematic attempt to identify which cellular functions are regulated either by Fn fiber rigidity, or by stretch-sensitive protein conformations. Here, we differentiate between these two causes by comparing cellular responses to cell-derived 3D scaffolds, and to one-dimensional Fn fibers in a stretched or relaxed conformation. Cell-derived matrices were used as our primary model system, since they are well characterized with respect to cell–matrix interactions.7,20,34,49,52,56,57 Moreover, FRET-labeled Fn, added to the culture medium, can be harvested by cells and incorporated into the ECM to directly report force-induced changes in Fn conformation.36,37 Thus the mechanical strain caused by cell traction forces can be mapped either in 3D cell-derived scaffolds or in the newly assembled Fn fibers. Chemically crosslinking of the scaffold allows us to address the differential roles of rigidity and Fn conformation, since it increases fiber rigidity while locking-in the more relaxed protein conformation of the de-cellularized scaffold. In the second part of our study we use a simplified, but more-controlled, model system using manually deposited Fn fibers with tunable rigidity and Fn conformation in order to dissect the causes for the observations made in the cell-derived ECMs. Chemical crosslinking has previously been employed to enhance the mechanical stabilization of tissue-derived scaffolds, yet with adverse clinical outcomes.13,14 We thus limited our studies here to chemical crosslinking, but later discuss the extension of our findings to crosslinking via natural biochemical pathways, as occurs in wound healing and diseases.
To grow cell-derived ECM scaffolds, NIH 3T3 fibroblasts were cultured for four days on glass and allowed to assemble soluble Fn into matrices that grew to approximately 17 μm thick. The 'four-day' matrices were then de-cellularized using an alkaline detergent solution. Similar to previous studies,20,56 the fibrillar structure of the de-cellularized matrices was preserved and their final thickness was about 10–15 μm. When needed, the de-cellularized matrices were subsequently crosslinked with 4% formaldehyde. There are numerous chemical compounds and non-chemical methods that have been used to crosslink tissue engineering scaffolds for medical applications,58 including formaldehyde and glutaraldehyde (e.g. ref. 13). Both chemicals have been used previously to study the effects of ECM rigidity on cell behavior49,56 and both increase Fn matrix rigidity by similar amounts;34 however, glutaraldehyde is autofluorescent. Therefore, formaldehyde was used in our studies as a model crosslinking chemical.
The native and crosslinked de-cellularized ECM scaffolds were reseeded with fibroblasts and imaged over 48 h to study how they were remodeled, and served as a framework for the assembly of new matrix. Since cells can harvest Fn from the cell culture medium and incorporate it into their own ECM fibers, adding trace amounts of donor/acceptor-labeled Fn (Fn-DA) allows live-cell imaging of the Fn matrix dynamics37,42 and mapping of the corresponding mechanical strains.38 To probe the mechanical reciprocity between old and new matrix, the cells were allowed to incorporate trace amounts of Fn-DA either into the 3D scaffold or the newly assembled matrix and the live cell cultures were then spectro-scopically analyzed. Due to the large size of Fn, the best mechano-sensitivity was obtained when Fn was labeled with multiple fluorophores: the four buried cysteines of the Fn dimer (modules FnIII7 and FnIII15) were labeled with acceptors, and approximately seven donors were conjugated randomly to lysines (Fig. 2a).37 Dilution of Fn-DA with an excess of unlabeled Fn prevented intermolecular FRET.37
Reseeded fibroblasts invaded the scaffolds by pushing fibers near the cell perimeter outwards and fibers underneath the cell downwards (Fig. 1a). The fibers surrounding the invading cells were progressively stretched and, in the native scaffolds, were compacted to form transient fibrous ring structures (arrows). Regardless of crosslinking, the cells typically resided (Fig. 1b) near the top of the scaffold, with the nucleus and sometimes part of the cell body exposed (cells 1 and 3) or near the bottom of the matrix with some parts of its ventral side or pseudopodia touching the surface (cell 2). The position of the cell in the scaffold did not appear to alter its morphology or behavior.
After invading the scaffolds, the cells began to migrate by grabbing distant matrix fibers with their pseudopodia and pulling their body forward while at the same time deforming the anchoring fibers. Obstructing fibers were pushed out of the way to accommodate the cell body (Fig. 1c and Supplementary Movie 1†). Once the cells had passed, the local matrix fiber deformations relaxed back, although not necessarily to the same position. When the fibers in front of the cell body were parallel to the direction of movement, there was little permanent fiber rearrangement (Supplementary Movie 2†). These behaviors were not altered in crosslinked scaffolds, although overall fiber movement was reduced (Supplementary Movie 3†). The presence of broad-spectrum protease and matrix metalloproteinase inhibitors did not affect the mode of cell migration (data not shown).59
While in situ force sensors have been available for several years,32,35,60 it is important to recognize that it is not the force that directly defines whether a molecular binding site is altered, but the molecular structure as distorted by tensile forces (mechanical strain). The advantage of FRET is that it directly probes the average increase of donor–acceptor distances and is thus sensitive to mechanical strain.37 Since the acceptors are located on modules FnIII7 and FnIII15, it is important to note that we were probing only the force-induced structural changes in the proximity (<12 nm) of these acceptors, as indicated by yellow spheres in Fig. 2a. The peak intensities of the acceptor and donor fluorophores were measured using spectral confocal microscopy (not correcting for residual cross-talk between the excitation and emission channels). The resulting intensity ratios, IA/ID, of the ECM fibrils are given as histograms (Fig. 2d and f), or spatially resolved as false-colored images (Fig. 2b and c). In order to associate the intensity ratios derived from ECM fibrils to structural alterations, they were compared to those obtained from soluble Fn-DA denatured in known concentrations of guanidine HCl (GdnHCl),37 or from individual Fn fibers in conjunction with stress–strain measurements (Fig. S1 in the ESI† and ref. 55). Previous work has demonstrated that Fn in solution begins to lose its secondary structure in GdnHCl concentrations higher than 1 M, and is completely denatured in 4 M GdnHCl (ref. 37). Moreover, the intensity ratio measured at 1 M GdnHCl correlates well with the intensity ratio below which we see a large increase of Fn unfolding in single freely suspended Fn fibers (approximately 150–200% strain).55 The 1 M GdnHCl denaturation curve point, which roughly corresponds to 200% strain (three-fold extension), was measured using Fn monomers in order to prevent additional FRET caused by crossover of the dimer arms.
Four-day ECMs, labeled with Fn-DA, were prepared, imaged before and after de-cellularization, and then 18 and 48 h after reseeding in order to map force-induced local alterations of Fn's structure. Representative slices from the confocal z-stacks of four-day and de-cellularized scaffolds are shown in Fig. 2b and c. There was no statistically significant difference in the intensity ratio between slices taken at different z-positions (data not shown).
Four-day matrices had an average median intensity ratio of 0.52 that shifted up to 0.57 upon de-cellularization. De-celullarization eliminated cell-generated matrix tension and thus allowed the ECM fibers to partially contract, as indicated by FRET recovery. The associated increase in FRET intensity ratio is clearly seen in the color shift from green-blue to yellow in the images (Fig. 2b and c) and in the right-shift of the corresponding histograms (Fig. 2d and f). FRET recovery was, however, not complete, indicating that the refolding of at least a fraction of Fn molecules might be inhibited by interactions with other tightly bound proteins. The de-cellularization procedure occasionally caused the matrix to tear or partially detach from the substrate. Damaged matrices were detected by visual inspection and by their very high FRET,37 and were excluded from the analysis.
To estimate the partial loss of secondary structure that occurs in the proximity of the acceptor sites, the percentage of pixels in the FRET histograms with intensity ratios less than those obtained for the Fn monomer in 1 M GdnHCl solution is shown as shaded regions (Fig. 2d and f), and is summarized for multiple experiments in the bar graph (Fig. 2e). Elsewhere, we show by straining freely suspended single Fn fibers, that massive exposure of the cryptic cysteines buried in modules FnIII7 and FnIII15 begins at a strain of 150% (ref. 55). The FRET ratios measured at this point on single Fn fibers correlated with those obtained for soluble monomeric Fn molecules in 1 M GdnHCl solution.55 De-cellularization of four-day old cell-derived matrices caused the percentage of fibers with perturbed secondary structure, to decrease from 82 to 61% (p < 0.05; Fig. 2e). These results agree with previous studies showing that the elimination of cell-generated tensile forces leads to partial matrix relaxation.37,42,61
Rigidifying Fn-based ECMs via chemical crosslinking affects cell migration,56 causes changes in the molecular composition of cell adhesion sites49 and increases the force necessary to detach cells from the matrix.34 We therefore asked if native and crosslinked scaffolds would experience differences in the time-course of remodeling. During the 48 h after reseeding, the fibroblasts progressively stretched the native scaffold fibers, which can be seen graphically in the change of the color-coded images in Fig. 2c and 3a,b from yellow-green (0 h) to green-blue (48 h) (corresponding histograms and controls can be found in the ESI, Fig. S2 and S3†). FRET revealed, however, that crosslinked scaffolds were not stretched during the 48 h of cell-mediated remodeling (Fig. 2c and 3c,d). While cells increased the percentage of partially unfolded fibers in native scaffolds from roughly 61 to 91% over the course of 48 h (p < 0.05), the strain of crosslinked scaffolds was not significantly altered (64% at 48 h, Fig. 3i).
To gain insight into how the alterations of the FRET ratios related to the average extensions of the ECM fibers, corresponding uniaxial, single fiber strains are shown on the far right side of Fig. 3i ("Single Fiber Measurements"). This FRET–strain correlation was deduced from a separate experiment using freely suspended Fn fibers.55 Crosslinking thus locked-in a more relaxed conformation of fibrillar Fn, corresponding to an average strain of around 200% that remained unchanged over time. In contrast, the Fn fibers of the native scaffold were stretched from about 200 to 300% strain over 48 h.
Because newly deposited matrix plays a prominent role in defining the properties of the cellular microenvironment,62 we asked how crosslinking the scaffolds would affect the deposition and conformation of new Fn fibers. Once seeded, the fibroblasts assembled new (Fn-DA-labeled) fibers throughout the full thickness of (unlabeled) native and crosslinked scaffolds. Similar to previous studies,42,56 the new fibers deposited on native scaffolds closely followed and co-localized with the existing matrix fibers, although some non-overlapping fibers were also observed (Fig. 4a–c, arrows). In contrast, when the scaffolds were crosslinked, the new fibers did not co-localize with the existing fibers, although they were anchored to them and aligned in the same general direction (Fig. 4e–g).
Significantly, the newly deposited Fn fibers in the native scaffolds, which were initially soft, showed little unfolding (approximately 100% strain, Fig. 3e and i), but were slowly stretched over 48 h as the cells actively remodeled their environment. From 18 to 48 h, the percentage of Fn with partial unfolding, as deduced from the FRET histograms, increased from 17 to 46% (Fig. 3i). Crosslinking of the scaffold had a major impact on the physical state of newly assembled ECM: the new Fn fibers deposited in crosslinked scaffolds were already highly stretched after 18 h with little further stretching as time progressed (Fig. 3g and h). They exhibited percent-partial-unfolding levels similar to those found in new ECM deposited by fibroblasts directly on glass (Fig. 3i). Thus, Fn fibers assembled by fibroblasts on crosslinked scaffolds were initially more unfolded than on native scaffolds, but by 48 h, they exhibited similar conformations. Based on previous work showing that cells on rigid 2D substrates generate more highly stretched Fn fibers than on soft 2D surfaces42, our results suggest that—at least initially— the cells in the crosslinked 3D scaffold were experiencing a far more rigid microenvironment and respond to it by increasingthe stretching of new ECM.
Earlier studies have shown that chemical crosslinking increases ECM rigidity enough to change cell behavior,49,56 and that cells can sense a wide range of elastic moduli.4,24,63 A study has recently used AFM nano-indentation to measure the increase in overall ECM rigidity due to crosslinking;34 however, we were interested in measuring the mechanical properties of individual Fn fibers to better understand how chemical crosslinking impacts the quantitative relationship between stress–strain characteristics and functionality. Because the cell-derived scaffolds had a broad distribution of thicknesses and spatiotemporal variations in strain, we performed our measurements on isolated, manually produced fibers that were more controlled with regard to these variables. We manually pulled Fn fibers from a droplet of concentrated Fn solution.38 The fibers were deposited across and attached to microfabricated PDMS ridges, and then rehydrated to enable force-extension measurements on freely suspended fibers in physiological buffer (Fig. 5a). Pulling Fn fibers out of a droplet requires > 10 μN of force and results in fibers with 140% pre-strain.55 Therefore, in order to relax the fibers to their fully relaxed state, the fibers were deposited on PDMS ridges that were pre-strained in a one-dimensional strain device; then the PDMS ridges, along with the fibers, were relaxed until fiber buckling was observed. Multiple, independent stress–strain measurements were performed on single fibers before and after crosslinking with 4% formaldehyde using a MEMS actuator and force sensor.64 As the tip advanced, the MEMS device measured the force acting on the tip while the fiber length and fiber diameter were monitored by confocal microscopy (Fig. 5b and c). The stress is the force normalized per unit cross-sectional area of the Fn fiber, and the fiber strain is given by (l−l0)/l0*100%, where l is the fiber length when stretched and l0 is length of the relaxed fiber. The Elastic or Young's Modulus is given by the slope of the stress–strain curve (see Materials and Methods). A detailed discussion of this technique is published elsewhere.55
Fig. 5d shows the force–strain curve for a representative fiber before and after crosslinking, as well as the stress (Fig. 5e) and Young's Modulus (Fig. 5f; data from additional fibers in the ESI, Fig. S4†). The force needed to strain the fibers was several-fold higher after crosslinking. For the representative fiber shown in Fig. 5, a strain of 80% required a force of 1.7 μN, whereas after crosslinking, a force of 5.5 μN was required (Fig. 5d). While Fig. 5 shows that the crosslinked Fn fibers are extensible, cell-generated forces did not seem to be sufficient to stretch them within the sensitivity of our FRET technique (Fig. 3). Note that the force needed to stretch a fiber is proportional to its cross-sectional area, which was approximately 7 μm2 for our manually pulled Fn fibers and therefore much larger than that of cell-made fibers. For example, the thinnest known Fn fibers are 10–12 nm in diameter65,66 and would only require 19–27 pN of force to achieve an 80% strain. While cells typically apply forces of a few nN per square micrometer of adhesion area,67 the forces cells apply to adhesions smaller than 1 μm2 can be considerably higher.32
The slope of the stress–strain curves for Fn fibers is not constant, unlike many linear elastic materials whose elasticity can be approximated by a single elastic spring model (Hooke's law). The Young's Modulus increases steadily as the fibers are stretched (Fig. 5f). Crosslinking the fibers also increased their rigidity (Young's Modulus) from 0.8 to 2.0 MPa for 0–50% strain (median values; p < 0.01, n = 8, paired t-test). For crosslinked fibers, only strains up to 200% are shown because these rigid fibers detached from the ridges under high force prior to their breakage. Taken with our earlier finding that cells are not able to cause detectable conformational changes in crosslinked scaffolds, these data imply that cell-generated forces are not high enough to stretch the fibers sufficiently to cause a detectable change in Fn conformation.
Does crosslinking affect the binding of proteins to Fn fibers and the mechanosensitive exposure of cryptic sites upon stretching? In particular, the cryptic self-association site located in Fn module III1 is known to be exposed by cell-generated strain and is involved in fibrillogenesis.46,68 We therefore asked if soluble Fn would bind to Fn fibers in a conformation-dependent manner and whether chemical crosslinking of the pre-strained fibers would affect this process. Again, because of the inherent variability of Fn fibers in cell-derived scaffolds, we used manually deposited fibers with controlled levels of strain.
Fn fibers containing Fn-DA were deposited in an asterisk pattern on a thin silicone sheet so that when one-dimensional strain was applied, a range of conformations was obtained (Fig. 6b and e).38 The stretched fibers were then either crosslinked or used in their native state. After incubating the fibers with soluble Fn-Cy5, the intensities of the bound Fn-Cy5 were measured at each pixel (Fig. 6c and f). Fig. 6a and d show Fn-Cy5 intensities on fibers with different conformations and demonstrate that the binding of soluble Fn increased with fiber strain, regardless of crosslinking. Nevertheless, our previous experiments showed that cell-generated forces were not able to stretch crosslinked Fn fibers sufficient to change their conformation, resulting in less partial unfolding in the crosslinked scaffolds (Fig. 3i). Taken together, our data imply that the binding of soluble Fn to fibrillar Fn is up-regulated in native but not in crosslinked scaffolds due to the limited force cells can apply to matrix fibers.
In our earlier experiments, the crosslinked cell-derived scaffolds provided a 3D microenvironment with relatively constant high rigidity and relaxed Fn conformation, while the rigidity and level of Fn unfolding in the native scaffold began low and increased over time (Fig. 3i). Although we hypothesized that the difference in conformation of the Fn matrix, newly deposited on the native and crosslinked scaffolds (Fig. 3e, g and i), was an effect of the elevated rigidity of the crosslinked scaffold, we could not exclude the possibility that it was an effect of a difference in the scaffolds' Fn conformation. This could be due, for example, to structural templating or due to a Fn-conformation-dependent change in outside-in cell signaling. In order to determine if the Fn conformation in existing fibers affected the conformation of newly deposited fibers, we studied the matrix assembled by cells seeded onto single Fn fibers with an adjustable conformation and constant rigidity. The manually deposited fibers provided a more controlled system than the 3D cell-derived scaffolds and, although the two model systems were different in many respects, a recent study has shown that cell migration in a one-dimensional environment mimics many of the characteristics of migration in a 3D environment.52
Individual Fn fibers were manually deposited on silicone sheets in a one-dimensional strain device, and either relaxed to an absolute strain of 56%or stretched to 500%(high unfolding; ESI, Fig. S1D†). The rigidities of single Fn fibers tightly physisorbed to silicone sheets were all in the range of a few MPa (Fig. 5f).55 Since it has been previously shown that NIH 3T3 cells are not sensitive to increases in the rigidity of 2D substrates beyond the order of 10 kPa,63,69 we conclude that the cells sensed a similar rigidity on the surface-attached native and crosslinked fibers.
After 18 h culture time, most of the cells remained solely in contact with the manually deposited fiber (Fig. 7a, i and ii) and had generated a Fn matrix (Fig. 7a, iii). Cells with pseudopodia contacting the BSA-blocked silicone surface were not analyzed. Cell-generated Fn fibers were attached to the manually deposited fiber (Fig. 7a, iv, arrows) and, at higher focal planes, wrapped around the cells (Fig. 7a, v, arrows). There was no statistical difference between the median intensity ratios of new matrix deposited on stretched and relaxed fibers (Fig. 7b). Moreover, there were no noticeable differences in cell or matrix morphology, and no significant differences in cell and matrix density between stretched and relaxed fibers (unpublished data). Taken with our earlier findings, this indicates that the cells primarily responded to the rigidity of the Fn fibers that were bound to the silicone sheets, rather than to the Fn conformation of stretched versus relaxed fibers.
Once cells are seeded onto a material, or into a 3D scaffold, they soon begin to assemble their own ECM, and will ultimately sense and respond to the new ECM fibers that surround them, and to which they are attached. While many studies have correlated how initial material properties correlate with cell fate at late time points,4,5,30,70 little information is available about how the properties of the microenvironment change over time. Seldom addressed is the role played by newly deposited matrix, much less how the properties of the new matrix are guided by those of the pre-existing matrix, scaffold or biomaterial. In an effort to shed light into the underpinning mechanisms, we made the following observations. Firstly, by applying local traction forces to the ECM, cells regulate the rigidity and biochemistry (Fn conformation) of their microenvironment in a time-resolved manner (Fig. 3, ,55 and and6).6). Secondly, the physical and biochemical characteristics of newly deposited Fn depend on the rigidity of the pre-existing Fn scaffold (Fig. 5 and and7).7). Thirdly, chemical crosslinking an ECM-based scaffold changes the time-course of the cell-mediated remodeling, not only of the scaffold, but also of the newly deposited Fn matrix (Fig. 3). Long-term cell fate might be impacted by the fact that traction forces cause a gradual increase in rigidity, which correlates with an increase in the average unfolding of Fn in both old and new ECM fibers. The stretching of fibers up-regulates the binding of soluble Fn (Fig. 6), which could at least in part be due to the exposure of cryptic sites on FnIII modules involved in regulating Fn fibrillogenesis.38,46,47,71 Thus, the timedependent changes in Fn conformation observed in the scaffolds and in the newly deposited matrix directly alter the physical as well as the biochemical properties of a cell's microenvironment, with the potential to switch outside-in cell signaling events.25,44,72 It has already been demonstrated that different conformations of surface-adsorbed, non-fibrillar Fn can impact cell differentiation.29,30,73
In light of the aforementioned studies that demonstrated adverse host responses associated with the crosslinking of tissue-derived scaffolds,13,14 we showed here that crosslinking of a cell-derived 3D scaffold increases the rigidity (Fig. 5f) and locks-in the conformation (Fig. 3i) of the scaffold, at least in the regime of forces that cells can apply to scaffold fibers. Most significantly, scaffold crosslinking profoundly affects specific characteristics of newly-assembled ECM. Crosslinking the cell-derived 3D scaffolds greatly accelerated the rate at which reseeded fibroblasts stretched and unfolded fibrillar Fn in their newly assembled matrix (Fig. 3i, closed squares), but decreased the amount of new matrix deposited (Fig. 3f and h). Using manually deposited Fn fibers with a controlled Fn conformation and constant rigidity, it was furthermore shown that the increased scaffold rigidity—not the locked-in, relaxed conformation—caused the reseeded cells to apply higher tensile forces at earlier time-points (Fig. 7b). The differences in the time-course of Fn unfolding between the native and crosslinked scaffolds, and the new matrix deposited therein, imply, as elaborated earlier, that cell-generated forces cause major differences in the physical and biochemical properties of new ECM fibers, which define the cellular microenvironment. Future research is needed to reveal more about the mechanisms of how ECM stretching might regulate certain outside-in cell signaling events and ultimately cell fate.48,74
Although it was beyond the scope of this study, naturally occurring ECM crosslinkers exist that alter cellular microenvironments during wound healing and in disease, and thereby direct cell phenotype. Crosslinks, regardless of their origin, cause an increase in mechanical stability. A recent study has demonstrated that the rigidity of Fn ECM is increased to a similar extent by treatment with formaldehyde, glutaraldehyde or factor XIIIa transglutaminase.34 Biological crosslinking enzymes such as the transglutaminases15 and lysl oxidase18 and naturally occurring crosslinks such as advanced glycation end products16,17 are associated with tissue stiffening in wound healing, and in diseases such as atherosclerosis and cancer. It is now well appreciated that microenvironment rigidity can guide cell phenotype. For example, the stiffening environment of the provisional matrix of a wound promotes differentiation of fibroblasts to myofibroblasts75 and a rigid ECM can promote a malignant phenotype.5 Future studies need to determine to what extent altered Fn deposition and strain-dependent alterations in conformation and rigidity play a role in determining cell phenotype in disease, and the maintenance of homeostasis.
In conclusion, we show for the first time to our knowledge that major time-dependent changes occur in the physical and biochemical properties of the cellular microenvironment, which consists of the 3D scaffold and the newly assembled matrix. The time-course by which newly assembled matrix changes its physical and biochemical properties, and the effects of scaffold properties need to be considered if one aims to decipher the molecular mechanisms by which microenvironment properties control cell phenotype. For example, the time required for an environmental stimulus (e.g. rigidity, Fn conformation) to determine cell lineage can vary between hours and weeks depending on the cell type and stimulus,4,29 yet how intermediate events regulate the late outcome is unknown. Proper consideration of how cell-generated forces alter the immediate cellular microenvironment as function of time is important not only in the design of tissue scaffolds but also to decipher how matrix biology directs long-term changes in cell and tissue phenotype.
Fn was isolated, labeled, and calibrated in GdnHCl (ESI, Fig. S1†) as previously described.37 Fn was isolated from human plasma (Zürcher Blutspendedienst SRK, Switzerland) by affinity chromatography. Fn was doubly labeled with Alexa Fluor 488 (donor) on amines and Alexa Fluor 546 (acceptor) on free sulfhydryls. The two batches of Fn-DA used in this study had an average of 7 donors and 3.5 acceptors per molecule. Singly labeled Fn was labeled on amines with Alexa 488, Alexa 546, Alexa 633 or Cy5. Labeled Fn was always used with an excess of 90–95% unlabeled Fn to prevent intermolecular energy transfer between molecules within a fiber. All dyes were from Molecular Probes (Invitrogen).
Manually deposited fibers were deposited on silicone sheets and strained on a custom-made, one-dimensional strain device as previously described.38 Briefly, 0.25 mm-thick silicone sheets (Specialty Manufacturing, Saginaw, MI) were cut into a dog-bone shape and cleaned by sonication in 2% PCC-54 (Sigma-Aldrich) and then 70% ethanol. A 300–500 μg ml−1 Fn solution (5% Fn-DA, 95% unlabeled) was deposited as a small drop on the silicone sheet. Fibers were pulled from the drop with a sharp tip and deposited onto the silicone sheet. Afterwards, the drop was aspirated and the sample washed with 2% (w/v) BSA in PBS, and soon thereafter immersed in PBS buffer for all further studies. Fiber strain was calculated from the macroscopic strain of the silicone sheet.37
For the binding assay of soluble Fn (Fn-Cy5) to stretched Fn fibers (Fn-DA), the Fn fibers deposited on silicone sheets were either crosslinked with 4% formaldehyde (EM grade; Polysciences, Inc.) in PBS for 1 h or used in their native state. After blocking with 4% BSA for 15 min., the fibers were incubated with 10 μg ml−1 Fn-Cy5 in 4% BSA in PBS for 20 min. at room temperature. After rinsing with PBS, donor, acceptor and Fn-Cy5 intensities were measured at each pixel. The Fn-Cy5 images were smoothed using a 3 × 3 pixel sliding averaging filter, and then the PMT background noise was subtracted. The Fn-Cy5 intensities were normalized to account for differences in protein mass by dividing each pixel's intensity by the sum of the peak donor and acceptor intensities (with 488 nm excitation) at the same pixel. This normalization method gives equivalent results to normalizing against the fluorescence of singly labeled Fn fibers (D.G., unpublished data).
Arrays of PDMS ridges were made using standard photolithography and PDMS replica molding. The master replica was then cast into a thin film of PDMS (1 : 10 curing agent to pre-polymer) on the aforementioned silicone sheets. The trenches between the ridges were 50–500 μm wide, about 20 μm deep and >400 μm long. To enable covalent crosslinking of the manually deposited fibers to the tops of the trenches, the structures were first treated with air plasma (250 mbar, 1 min.), then with 3% 3-aminopropyldimethyl-methoxysilane (ACROS Organics) in deionized water for 20 min, and finally with 0.5% glutaraldehyde (Sigma-Aldrich) in water for 30 min. Glutaraldehyde is a simple bi-functional crosslinker, so this procedure resulted in the glutaraldehyde binding with one aldehyde group to the PDMS ridges and presenting the other (active) group to allow covalent attachment of the Fn fiber. The substrates were rinsed with deionized water to remove unreacted glutaraldehyde and dried. The substrates were then pre-strained with the 1D strain device, and Fn fibers were manually deposited across the trenches. The parts of the Fn fibers in contact with the ridges became covalently linked to the PDMS via the free aldehyde groups. The samples were then relaxed until the first signs of fiber sagging into the microfabricated trenches were noticed, indicating that the fibers were fully relaxed. Since the fibers spanned multiple trenches, it was possible to measure each fiber independently in multiple locations, before and after crosslinking. Fibers were crosslinked with 4% formaldehyde for 1 h at room temperature, then incubated for 3 h in PBS before being measured again.
The 3-axis micromanipulator (Sutter MP-285) with an attached custom-made MEMS force sensor64 was mounted to the stage of a confocal microscope (Olympus FV1000; 40×, 0.90NA, water-immersion objective). The micromanipulator advanced the MEMS device at a constant velocity as controlled by a computer. Because the tip deffected in response to resistive force, it did not proceed with a constant velocity and thus fiber strain was measured from images taken with the confocal microscope. The force on the tip (F) was corrected to yield the tensile force on the fiber (f) using the following equation (x, extension).
The stress was calculated from the measured force values by dividing by the fiber's cross-sectional area, which was assumed to be constant over all measured strains.55 The cross-sectional area was measured from confocal images by averaging the full-width-at-half-maximum of the fiber measured at five different locations. The elastic modulus at a given strain was calculated by dividing the change in stress by the change in strain between the data points two-before it and two-after it.
NIH 3T3 fibroblasts (American Type Culture Collection, ATCC) were cultured in DMEM (ATCC) supplemented with 10% newborn calf serum (Gibco, Invitrogen) in a humidified incubator at 37 °C with 5% CO2. During experiments, the medium contained 1% penicillin–streptomycin–fungizone (Gibco, Invitrogen). The cells used for the production of the 3D ECMs were pre-conditioned to decrease their contact inhibition by culturing them for 20 passages as described in other studies.20,49 When needed, the cells were stained with the membrane-binding dye DiD (Molecular Probes, Invitrogen).
In order to prevent the cell-derived ECMs from detaching, glass surfaces were covalently functionalized with unlabeled Fn. 8-Well chambered coverglasses (Lab-Tek, Nalge Nunc) were treated with air plasma (PDC-32G, Harrick Scientific) at 250 mbar for 30 s. The surfaces were then treated with 2% 3-aminopropyldimethylmethoxysilane (ACROS Organics) in deionized water for 15 min. The surfaces were then incubated with 0.125% glutaraldehyde (Sigma-Aldrich) in water for 30 min. Finally, the surfaces were incubated with 20 μg ml−1 unlabeled Fn for 1 h at room temperature or overnight at 4 °C. The chambers were sterilized by UV irradiation for 15 min.
The cell-derived ECM procedure is based on previously published protocols.49,56,66 Cells were seeded on the prepared surfaces at 80 × 103 cells cm−2 and were allowed to attach for 30 min in the incubator; then the medium was replaced with medium containing 50 μg ml−1 Fn. The cells were incubated for four days, with one medium exchange after two days. On day four, the samples were washed twice with PBS, then three times with (0.1 M Na2HPO4, 2 mM MgCl2, 2 mM EGTA, in water, pH 9.6). The samples were then incubated with lysis buffer (0.5 M Na2HPO4, 1% Triton X-100, pH 9.6) at 37 °C. After 15 min, the lysis buffer was exchanged and the samples were incubated at 37 °C for 1 h. The samples were rinsed twice with (10 mM Na2HPO4, 0.3 M KCl, in water, pH 7.5), three times with deionized water, and once with PBS. Chemical crosslinking was performed immediately after de-cellularization. Matrices were incubated with 4% formaldehyde for 1 h at room temperature. The matrices were then washed four times with PBS and incubated for 3 h in PBS before cell reseeding.
Cell-derived ECMs were reseeded with 50 × 103 cells cm−2 for FRET experiments or with 6.25 × 103 cells cm−2 for time-lapse experiments and incubated for 30 min in the incubator; then the medium was replaced with medium containing 50 μg ml−1 Fn. For inhibition of extracellular proteolysis, the sample medium additionally contained 20 μM Ilomastat (Chemicon, GM6001) and a 1 : 400 dilution of a protease inhibitor cocktail (Sigma, P1860) containing Bestatin, Leupeptin, Aprotinin, Pepstatin and E64. An equivalent volume of DMSO was used as a vehicle control.
Fn fibers containing Fn-633 were manually deposited on silicone sheets either parallel or perpendicular to the direction of strain, and strained as described in the main text. The samples were blocked in 2% BSA for 1 h at room temperature. Cells were seeded onto the samples and incubated for 15 min to allow them to attach, then rinsed with medium to remove unbound cells. The samples were incubated for 30 min; then the medium was replaced with medium containing 2% BSA, 45 μg ml−1 unlabeled Fn and 5 μg ml−1 Fn-DA. The samples were cultured for 18 h, then fixed with 4% formaldehyde and imaged.
Z-stack images were acquired with an Olympus FV1000 confocal microscope with an oil-immersion 1.35NA 60× objective. Our basic acquisition procedures for FRET imaging have been described previously.37 For time-lapse microscopy, the confocal microscope was fitted with temperature and CO2 control chambers (Life Imaging Services, Switzerland) that maintained the entire microscope at 37 °C, and saturated the sample with humidified air containing 5% CO2. Images were taken every 20–30 min. for approximately 16 h.
The acceptor and donor images were analyzed using MATLAB (MathWorks, Inc.) as previously described.37 Briefly, after correcting for the background noise and differences in the scaling of the PMTs, the acceptor intensities were divided, pixel-by-pixel, by the donor intensities to yield the intensity ratio. All z-slices were processed individually.
Glass surfaces functionalized with silane and glutaraldehyde had a high background fluorescence that skewed the FRET ratios of fibers within about 2 μm of the surface to higher values. In the absence of surface functionalization, all z-slices in a z-stack had approximately the same intensity ratio distribution. Therefore, slices near the functionalized glass surface that showed an increase of more than 0.01 intensity ratio units were automatically detected and removed from further analysis.
Some images from four-day ECMs were contaminated with signal from endocytosed fluorescently labeled Fn that appeared in small, round vesicles as previously observed.76 The FRET ratio of endocytosed Fn-DA centered near 0.2 and was well-separated from that of matrix fibers. Large regions of the contaminating signal were removed directly from the images by hand. Histograms of the data from images thus corrected were not significantly different from histograms of compiled data from images of similar samples not containing endocytosed Fn-DA signal.
The crosslinked scaffolds contained small (<3 μm) particulate debris that adsorbed Fn-DA, and contaminated the 18 and 48 h reseeded FRET images. Spots that had an area <100 pixels were automatically removed from these images. This algorithm had no significant effect on the median intensity ratio when applied to FRET images of matrix without particulate debris. All of these corrections were only implemented for data analysis; the images shown in this paper were not altered.
Statistical analyses were performed with MATLAB (MathWorks, Inc.) and SPSS (SPSS, Inc.). Percent unfolding and median intensity ratios for the cell-derived ECM scaffold experiments were compared using a one-way ANOVA, followed by Tukey's multi-comparison test. Unless otherwise stated, errors and error bars indicate standard deviation.
Cell and tissue fate is intimately linked to the biochemistry and physical properties of their microenvironments (including mesenchyme, stroma and stem cell niches), yet no tools were available to probe how cell-generated tensile forces physically alter the properties of 3D extracellular matrix. FRET-labeled fibronectin is exploited as a mechanical strain sensor to monitor time-dependent changes of either tissue-mimetic 3D, ECM scaffolds reseeded with cells or of the newly assembled matrix fibrils. This in situ method is versatile, since cells, provided exogenously with FRET-fibronectin, typically co-assembled it into their ECM fibers. Probing how environmental conditions regulate cell-mediated stretching of ECM fibers is physiologically significant because stretching alters fiber rigidity and the presentation of molecular and cellular recognition sites (mechanotransduction).
We thank Sheila Luna for designing the cartoon in Fig. 5b, assistance with rendering the 3D reconstructions in Fig. 1, and the isolation of Fn. We also thank Li Bojun for measuring the silicone sheet stiffness. This project was financially supported as part of the Nanotechnology Center for Mechanics in Regenerative Medicine (an NIH Roadmap Nanomedicine Development Center), the Volkswagen Stiftung, and the ETH Zurich. MLS was funded by the Human Frontier Science Program Organization.
†Electronic supplementary information (ESI) available: Supplementary figures and movies. See DOI: 10.1039/b914996a
Conflict of interest statement: The authors declare no competing financial interests.
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.


text:
Getting a spa treatment is a great way to reward yourself, much like presenting yourself with a check for $1,000 worth of self-hugs (one). Thank you with this voucher.
Choose Between Two Options
- $99 for six ultrasonic-cavitation treatments (a $1,500 value)
- $129 for nine ultrasonic-cavitation treatments (a $2,250 value)
Machinery emits low-frequency sound waves to diminish fat cells during noninvasive treatment sessions.
Intuitive Skin Studio
The skin therapists of Intuitive Skin Studio rely on advanced procedures to fight the signs of aging: HydraFacial diamond microdermabrasion, LED photo facials, radio frequency facials, micro-needling dermapen treatments, and LHE phototherapy. Every treatment includes a complimentary consultation, and all sessions take place in a private treatment room. Meanwhile, massage therapists pamper clients with relaxing, therapeutic sessions, employing Swedish, deep-tissue, and trigger-point techniques.


text: Hi guys.. I have bought an Inspire 1.. I have setted the tablet android with the remote, leaving the inspire in the carrying case because I thought it's safe.. But.. I have accidentally disabled travel mode and the inspire starts to lower the landing gear... Obviously has forced in the bag, the foam is very hard to move.. But the inspire seems ok.. I've tried a couple of time uppering and lowering the gear and it's ok.. But..... When the gear is up (flying mode) the servo make a noise, like the gear is not up at the maximum position.. I hear that noise when I have the inspire in my hand.. Long story short... Anyone has make the same thing happened to me? Yours inspire make a noise when in your hands with the gear up? Thank you..


text:
ReportSee more
Smoking [Solved]
Ask a question FeaLaep 1Posts Thursday August 29, 2013Registration date August 29, 2013 Last seen - Last answered on Sep 28, 2013 at 01:33 AM by raja.rickly
Member requests are more likely to be responded to.
Members can monitor the statuses of their requests from their account pages.
A CCM membership gives you access to additional options.


text:
25 Craves of Christmas
Dec. 5: Christmas Donuts
The snow is coming! So spend the day in your PJs and treat yourself to these decadent donuts--the perfect pastry to keep your mind off of the cold weather. Kick back, relax, and enjoy! (Courtesy of Taste of Home)
- 6 Eggland's Best Eggs
- 2 cup sugar
- 1/4 cup canola oil
- 1 teaspoon lemon extract
- 7-1/2 cups all-purpose flour
- 6 teaspoons baking powder
- 2 teaspoons salt
- 1 teaspoon ground nutmeg
- 1 cup milk
- Additional oil for deep-fat frying
- Confectioners' sugar, optional
- In a large bowl, beat the eggs, sugar and oil until blended. Beat in extract. Combine the flour, baking powder, salt and nutmeg; stir into egg mixture alternately with milk. Cover and refrigerate overnight.
- Divide dough in half. Roll out one portion to 1/4-in. thickness. Cut with a floured 1-1/2-in. round cookie cutter. Repeat with remaining dough.
- In an electric skillet or deep-fryer, heat oil to 375°. Fry doughnuts for about 4 minutes or until golden brown, turning once with a slotted spoon. Drain on paper towels. Dust with confectioners' sugar if desired. Yield: about 8 dozen.


text:
Some people date for the long run while other date for the fun of it. Some people, like me, date for marriage. Others want one night stands. So what kind of dater are you? Do you brag about how many people you've dated or how wonderful your last relationship was?
Most Helpful Girl
Quality over quantity definitely. It's better to date people who have qualities that match yours. How many people you date isn't important it's the nature of the relationship that counts00|00|0


text:
- Description
The contrasting microfiber and bi-cast vinyl covering of the Anthony Collection highlight this contemporary seating group.
Pocket-coil seat cushions provide a comfortable living space for your family and your friends.
Extensive assemble may be required.
More from this collection: Anthony
- Additional Information
Additional Information
SKU 13401691 MPN 9904-1 GTIN UPC 782359136417 Brand Homelegance Collection Anthony Assemble Length 34 Assemble Width 33 Assemble Height 34.5 Assembly Required Yes Material Microfiber Color Brown Styles Transitional Shipping Time 1-2 Weeks Free Shipping Yes
- Reviews
Disclaimer: Pictures displayed may include optional products not included in the price. Computer monitors often display color variations. Color swatches are shown if alternative colors are available. Please call us or email us if you have any questions!


text:
> Shoes
> Pumps
> New Fashion Sexy Women Plus Size Big Bow Pointed High Heel Stilettos Shoes Pumps Wedding Party Evening Shoes
Material: Synthetic Leather
Sole Material: Rubber
5 Colors available: Red, Pink, Green, Black, Blue
Type: Pumps
Shoe Style: Two-Piece Style
Heel Height: 11cm/4.3″
Heel Type: Thin Heels
Toe Shape: Pointed Toe
Shoe Width: Medium
Decoration: Bow
Pattern: Solid
Occasion: Casual, Party, Wedding, Evening Party
Season: All Season
Open Type: Velcro
5 beautiful and bright colors for your choice
Comfortable material
With decor of a big bow
This Shoes is very fashion and popular!
Popular design, we believe you will love it very much!
Join our community
Subscribe To Our Newsletter:Subscribe
Copyright © 2015-2016 Dressgal. All Rights Reserved.


text:
Author Tags: Fiction, Poetry
Sandra Janssen is a writer of literary fiction and a publisher. Writing under the name of Elizabeth Gallacher she has published three novels - Circle of Beginning, September Gales and Circle of Attachment. She will shortly publish Circle of Intent.
Born on Vancouver Island she spent over a decade as a special needs educator before going to the UK to work for Harvard University Press, the MIT Press and Pluto Press. She has also worked in H.K. Lewis & Sons Medical and Scientific bookstore and for Cambridge Medical Books, a UK library supplier.
While in the UK she taught a number of creative writing, creative recovery and book marketing courses and workshops. She has won a number of awards for her poetry and short fiction in the UK and Canada. Her poetry has been published in the UK anthologies Summer Explosion and Winter Celebration.
She is also the owner of Windshift Press, which is now in its eleventh year of operation, having published over 40 titles in that time. She is currently working on the remaining volumes of the Circle series and looking forward to publishing Circle of Intent in early 2012.
Date and Place of Birth: Nanaimo, BC 24/06/48
Place of Residence: Vancouver Island, BC
Ancestral Background: Danish, Scottish
Circle of Beginning [BC: Windshift Press, 2005]
September Gales [BC: Windshift Press, 2011]
Circle of Attachment [BC: Windshift Press, 2011]
Summer Explosion [UK: Mast Publications, 1994]
Winter Celebration [UK: Mast Publications, 1995]


text:
Hollywood filmmaker Brett Ratner, who worked on Hrithik Roshan-starrer Kites, has shown interest in working with the Bollywood star once again, as he finds him to be one of the most happening people in India.
Ratner edited the English version of the film starring Roshan and Mexican actress Barbara Mori, and since then they share a strong friendship.
Roshan recently saw Ratner's Tower Heist and was left impressed.
He took to micro-blogging site Twitter to congratulate the filmmaker.


text:
What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
Editing product imagesAsked by seeku_joll on April 30, 2016 at 07:50 AM
Editing product images
JotForm Support
Hello seeku_joll,
If you're working on a form and would like to add images to your payment tool, uploading your image from a third party source. One of the common favorites is creating a Dropbox account and uploading your images there.
Once the images are uploaded, get the link to the image and proceed with your payment wizard. You will notice in the adding/editing of products there is a textbox for Image URL. You will place the link to the image in that field and then just save the products that you would like to have in your form.
Once complete the image should appear in your form.
Here is a sample animation provided by my colleague on how it should work:
If you have questions, please do let us know.


text:
Follow Us
An Alsace based winery.
By Nichoals Sterling
Hugel & Fils is one of the most traditional wineries of the Old Continent. Its history began in the 17th century, when the young Hans Ulrich Hugel settled in Riquewihr, a town in Alsace, France, on the border with Germany, which was disputed for centuries and at the time was completely devastated after the Thirty Years War.


text:
This is only a concept so far. He's a support champion.
Leonardo is a flamboyant painter.
(Passive) Magical brush: After casting a spell he may trace a line by left clicking and dragging the cursor around. This can be any shape. The starting point of the line must be within 600 range of Leonardo and none of the line can exceed 1200 range of him. He is immobile while drawing (he can still move around before starting to draw it however). Releasing left click will end the line and he will have to cast a spell before drawing another.
After finishing the line it appears momenterally to enemies before becoming invisible. Lines appear to allies all the time once it's finished. Only Leonardo can see the line while he is drawing it.
While ready to draw a line a range indicator is visible at all times on where he must start from.
His spells will cause the line he drew to cause an effect to the area it covers. And consume the line allowing him to draw another.
He may not cast any abilities until he has drawn a line.
Lines remain on the field until he casts a spell or dies.
The longer the line is the less powerful the effect. A line less than 100 will have it's full effect. Every 100' more will cause the effect to diminish by 10% to a minimum of 10% effectiveness.
If a spell is activated and it doesn't hit anything 80% of its mana cost is refunded and its cooldown is reduced to 2 seconds (to let you erase a line which you don't have use for, casting a spell you don't need).
(Q) Hell Brush: The area under the line erupts into flame for 2-5 seconds, dealing magic damage every second to enemies under it. The shorter the line is the more damage it deals. (10-5 second cooldown from when the fire fades)
(W) Soothing Brush: The line glows green for 1 second, healing allies that pass through it (only affects the same ally once) for a base amount plus a portion of their missing health. The base amount is lowered for a longer line. (10 second cooldown from when the line fades).
(E) Captivating brush: The line glows blue for 1 second, lowering the enemy's speed which pass over it for 2-5 seconds. The power of the slow is reduced for a longer line. It snares with the shortest line. (10 second cooldown from when the line fades).
(Ult) Inspirational masterpiece: The line flashes crimson, any allies which its over will receive a buff which increases their damage against enemy champions while decreasing the damage they take from them. This will cause a decrease in effectiveness not based on the length of the line rather than the amount of champions it affects (only affecting one champion will cause full effect, 2 champions will both recieve 10% less of the effect, 3 champions 20% and so on) so if you want to affect your entire team quickly scribble everywhere and cast.
While a support he can also be played as a burst mage, drawing a dot then hitting E to snare an enemy, then drawing another dot over them and hitting Q will cause a lot of damage to them.
Lines which backtrack over themselves don't count as being longer.
Have your team group together, unless the enemies have AoE, for bigger healing potential.
Use the maximum range to judge how long your line is. If you started your line at the very edge you were allowed to (600) and drew a straight line to the maximum range (1200) then your line is 600' long.
You may draw a new line when you cast a spell rather than when the line from the spell fades.


text:
|Property type
Property details
Brief description
Beach/water: River in approx. 4 km, Sandy beach in approx. 40 km
Bedroom/s: 1
Floor space: 60 m²
Next village/town: Caen in approx. 25 km, Center Curcy-sur-Orne in approx. 2 km
Number of persons: 2-4
No pets allowed
Room/s: 2
Type of property: house attached to another house
Detailed description
house attached to another house, 2 - 4 people, 2 room/s, 1 bedroom/s, 1 bathroom/s, approx. 60 m², 1 floor/s, access to the property via external stairs.
Equipment: satellite TV; radio; electric heating; storage room (external access only).
Feature: natural stone house.
Furnishing: agreeable, well-kept, tiled floor, laminate floor.
Room layout
Bedroom 1: (raised ground floor): queen-size bed (1.60 m wide).
Dining area: in the living room.
Important information: maximum occupancy: 2 adults and 2 child(ren).
Kitchen: Kitchen/ cooking equipment: 3 ceramic hobs, oven, microwave oven, dish-washer, combination fridge-freezer, coffee machine, water boiler, toaster. open to the living room.
Living room/bed room: (raised ground floor): double sofa bed.
Sanitary 1: (raised ground floor): shower, WC.
Estate: (shared use with the owner): approx. 10000 m², closed plot (fence, hedge, entrance gate), in a hillside, with plants, meadow. Owner lives on the property. Owner has horses.
Situation: rural.
Exterior (private use)
Plot description: furnishing provided, transportable barbecue. parking on the estate. private part of the estate approx. 600 m², open plot, in a hillside, with plants.
Beach/waters: River in approx. 4 km (Orne), Sandy beach in approx. 40 km (Ouistréham)
Next village/town: Caen in approx. 25 km, Center Curcy-sur-Orne in approx. 2 km
Shopping: Next shopping possibility (Thury-Harcourt) in approx. 4 km, Next supermarket in approx. 4 km
Paying per credit card incurs fees in the amount of 1% of the sum due.
*Plus compulsory or optional charges based on actual occupancy or consumption. The payment has to be made in the currency offered by the property manager, before use of conversion tool. Payments in other currencies have to be negotiated beforehand with the property manager. The currency conversion tool is provided for your convenience only and is non-binding.


text:
A motorcycle accident slowed northbound traffic on Texas Highway 195 just before 2:45 p.m. Thursday in Killeen.
The accident involved a vehicle and a motorcycle with a male and female rider.
Both the man and woman were transported from the scene by ambulance, witnesses said.
More on this story as it develops.


text:
This landlord has a strict landlord policy. Please read the Landlord Policies section carefully.
Please note that although the property has not yet been visited, Spotahome is in the process of verifying this apartment. Check back in a few months for a video guide, Spotahome photos, and personalized descriptions of the apartment!
This 6 bedroom, 2 bathroom apartment is located on Paseo de Santa María de la Cabeza in the residential and well-connected area of Delicias. The flat offers 6 fully and similarly furnished rooms with plenty of storage space; the shared areas include the bathrooms and the fully equipped kitchen with an oven. Situated to the south of the city center, you'll be within walking distance from the Atocha Train station and the lively, multicultural area of Lavapies. These rooms would be ideal for those looking to live in a comfortable home while having easy access to transportation lines.
The exterior apartment is located on the third floor of a building that is equipped with an elevator. Water, electricity, gas, Wi-Fi internet and cleaning are included. Wi-Fi is installed in the property and the cleaning is organized once a week.
Puerta de Atocha train station is just a few minutes walk away - from here you can catch trains to Valencia, Barcelona and plenty of other parts of Spain. You will also be around the corner from Palos de la Frontera station with yellow line 3.
Pets: not permitted
Couples: rooms which permit couples will have a rent increase of €85 per month when occupied by a couple
Utilities limit (Barcelona only): €90 per month
15-day booking policy:
Example: If the previous tenant leaves on July 1, you must move in by July 15
Fortnightly payment policy:
Documents required:
Check-in policies: (Madrid only)
Check-in policies: (Barcelona only)
Additional fees:
More specific landlord policies:
Suitable for: Professionals, Couples, Students, Erasmus
This part of the city, located near Puerta de Atocha, is perfect for those looking for an authentic Spanish neighborhood with relatively affordable prices in spite of it being close to so many tourist attractions. Not only is it ideal, but also well-connected to suburban university campuses (including Comillas campus in Alcobendas, Carlos III in Getafe, the Autónoma, and the Universidad de Alcalá de Henares), and also touristic spots like el Retiro, Tierno Galván park and the city's most reputable museums. It's location is perfect for services to other cities in Spain via Larga Distancia, Media Distancia and AVE high-speed trains and all the Cercanías commuter train lines.


text:
finite elements and two-phase flow ?
I am trying to simulate a - what I thought - rather simple test-problem. Consider a closed container, the lower half filled with water, the upper half with air. Gravity is acting downward. No walls are moving, so you would expect this configuration to be steady. Any simulation should give you zero velocities, nearly constant pressure in air and (with depth) increasing pressure in water.
Using a standard finite-element Galerkin method (Taylor- Hood triangle) in combination with the VOF-approach (linear approximation of density and viscosity, both fluids simulated), I found this problem can *not* be handled. During the first timesteps the velocities remain zero and the pressure solution is exact. Later the surface becomes unsteady and with increasing velocities the simulation "explodes".
Simulating the same problem without gravity results in no problems. The code without VOF has been validated in lots of problems including gravity-related ones. Computing other problems with the VOF-approach without gravity gives again no problems. The VOF-function is approximated by a linear function within the triangle.
Summing up I wonder whether there is some bug in my code or if this is some other (deeper) problem related to the linear VOF-approximation in combination with finite elements.
Does anyone have similar problems - or (even better) could hint at some inconsistency with the above approach ?
Any ideas are welcome ...
Thanks, Chris
Re: finite elements and two-phase flow ?
I am no expert in VOF, but it sounded like that when you turn on the gravity, the water is on the top and the air is on the bottom. Change the direction of your gravitational field to see whether your bugs are still there.
Re: finite elements and two-phase flow ?
Thanks for responding John. This was of course one of the first things I tested when I first saw the problem. Unfortunately the whatever-it-is-problem lies somewhat deeper.
|All times are GMT -4. The time now is 06:21.


text:
By Anne E. Kornblut
Thursday, April 22, 2010; C01
BARCELONA -- Janet Napolitano stood near the water's edge, arms crossed, gazing up at a giant X-ray contraption.
It was a sleepy Saturday in Barcelona, and the city port was deserted. But the secretary of homeland security, who had flown in for a few hours on her way to Nigeria, was confronting this elephantine tangle of metal. This stop was the second on an international tour she launched after an attempted airliner attack on Christmas nearly became the Obama administration's Sept. 11, 2001.
Surrounded by rows of shipping containers, 42,000 of which are sent to the United States each year, Napolitano quickly summed up the magnitude of her task, which even a machine this large could hardly address.
"Is there only the one scanner for the entire port?" Napolitano asked.
Yes, her guide replied. Napolitano winced.
A moment later, local officials started up the X-ray machine for her. It malfunctioned. "This doesn't make any sense," she fumed. "I've watched these scanners all over the place. Why doesn't this one work?"
That question -- of whether security devices work -- is one that has haunted Napolitano ever since a 23-year-old Nigerian named Umar Farouk Abdulmutallab tried to down Northwest Airlines Flight 253 in Detroit. After the incident, Napolitano's misconstrued claim that the "system worked" became a national punch line, one that briefly seemed as if it might derail her upward trajectory.
But four months later, after hunkering down in the granular details of international aviation standards, Napolitano has reemerged as one of the strongest members of the Obama Cabinet: On the shortlist for the Supreme Court, in line if the attorney general position should become available and, just this week, front and center at the Oklahoma City bombing memorial.
Senior administration officials describe her as one of the most astute members of the national security team, some in hyperbolic terms. White House Chief of Staff Rahm Emanuel, in a recent interview, declared himself as "head over heels for her," which doesn't happen often. White House terrorism adviser John O. Brennan hails her as "passionate" and "formidable."
How Napolitano, 52, won over hard-to-please heavyweights while managing the most unwieldy department in Washington is a testament to her relentless persona. The tough and stocky former prosecutor once climbed Mount Kilimanjaro, and even delivered her speech at the 2000 Democratic convention three weeks after a mastectomy. Napolitano embraced the Christmas Day bomber crisis as an opportunity -- to the surprise of no one close to her -- and told President Obama she would use it to seek cooperation on airline security from recalcitrant countries. To this day, she chipperly describes the near-disaster as a "catalyst" for improvements.
In the months since, she has hopped from Japan to Mexico to scout the global aviation mess. She worked her way, Captain Ahab-style, toward Nigeria, the country where Abdulmutallab began his journey. Yet as Napolitano flew into the capital Abuja in mid-April, just hours after reviewing the dockside headaches of Barcelona, she came face to face with the dangers that make global travel so impossible to police. She would spend just 24 hours in Nigeria, traveling under heavy security in a city so violent her contingent was advised not to leave the hotel. Nonetheless, her goal was to somehow grasp the airport security status of the entire African continent, in the hopes of stopping a single terrorist from ever slipping lethal materials onto a U.S.-bound plane again.
Under the circumstances, Napolitano sounded surprisingly philosophical. "Something could always get through," she said, leaning forward in her seat aboard the small military plane she uses for travel.
"We live in a world where we don't provide guarantees. We provide the ability to identify and minimize risk, and to respond quickly should a risk materialize," she said, in a low, serious voice. "But if something happens in the United States, we also have to have confidence as a people that we'll be able to respond."
Does she ever feel panicked about the impossible number of threats? "No," she replied, without hesitation.
Told that some of her aides describe her as unflappable, almost Zen, under pressure, she did a mock double take.
"Did you say 'thin?' " she said, laughing.Taking the 3 a.m. call
Napolitano's wry sense of humor is a defining trait. She also has an encyclopedic knowledge of pop culture, able to spend hours discussing movies and television ("The Wire" and "Mad Men" are particular obsessions). Oddly, given her role, the only series Napolitano seems not to watch is terrorism thriller "24" on Fox. But then, she does not have to.
"Remember in the presidential campaign, the 3 a.m. ad?" said Sen. Mark Warner (D-Va.), a friend of hers, referring to the Clinton campaign's provocative questioning of Obama's ability to handle a crisis. "The secretary of homeland security is actually the person who has to take that 3 a.m. call. Even when it is a false alarm."
When the 3 a.m. call came on Christmas Day, it came after breakfast. Napolitano was at her brother's house when news broke that a young Nigerian had detonated an explosive device hidden in his underwear during the flight's approach. She joined in flurries of phone calls, learning that the suspect had boarded with a valid visa but no extra screening despite red flags.
The following morning, while Obama and other counterterror officials stayed on vacation, Napolitano was dispatched to the television networks. On two Sunday shows, she sounded firm, insisting that, once the bomb failed to detonate, authorities had things under control. But on a third, she truncated her talking point to declare "the system worked" -- triggering a wave of criticism, especially from Republicans who ridiculed her for failing to grasp what had failed.
Within the White House, her support never waned despite the unfortunate choice of words. "Anybody in public service goes through rough spots," Emanuel said. "The question is, (a) do you learn something? And (b) how do you handle it when you go through it?" Several other officials said she had been right where it mattered -- in her actions, if not her verbiage. Tom Ridge, the first DHS secretary, said she had been unfairly criticized.
Napolitano is matter-of-fact about the incident, if not exactly thrilled to discuss it. "There's no time to sit and moan about a quote that's something you said," she said. "You've just got to move."
If further proof of her resilience were needed, it came earlier this month, as Napolitano traveled thousands of miles from Washington. Word leaked that Justice John Paul Stevens would retire from the Supreme Court after this term -- and that Napolitano, whom Obama interviewed during the last vacancy, was again on the shortlist.
A former popular governor of Arizona elected during an era of Republican dominance in 2002, Napolitano would bring to the court the kind of "real world experience" Obama said he prefers. And there is no question she would want the job. Described by friends as a striver with higher aims, she has been discussed as not just a justice or attorney general but perhaps even a presidential candidate.
Still, traveling abroad as speculation about her future buzzed, Napolitano stuck to her script. She was flattered, but focused on her job, she said. Only during a long flight did she let her guard down, allowing that it was an odd sensation to be talked about as a potential justice.
"It doesn't, in a way, seem real, because you're reading about yourself. And I'm Janet," she said. "That seems like it's going on in another world, in a way. It's very unreal in that sense. And it is flattering. Who wouldn't be flattered by that?"A 'hard-charging boss'
More to the point: What secretary of homeland security wouldn't eventually want an escape hatch?
It is a thankless job, one that puts its occupants in the spotlight only when disaster strikes. When she hasn't been managing terrorist threats, Napolitano has worked on other nightmares -- the H1N1 flu crisis, border violence. It is a "24/7-type job," as she describes it, just as being governor was, meaning that she has basically been on the clock for the past eight years.
Even during a lull, she is in motion. Phone calls to her staff run as late as 11:45 p.m., sometimes with another at 5:30 a.m. (she does not do e-mail, and only recently got a cellphone). Around headquarters at the Nebraska Avenue Complex, not far from her condo, Napolitano has been known to confront aides about their missteps. Her chief of staff, Noah Kroloff, 37, who has worked for her for a decade, describes her as an "incredibly hard-charging boss."
That may be the only kind of boss DHS requires. The third-largest department in the government, cobbled together with 22 agencies after Sept. 11, 2001, it is still an impenetrable alphabet soup of regulations. DHS has so many acronyms that there is now an iPhone guide. (What other Cabinet secretary has an app for that?)
Brennan, the White House official who works most closely with Napolitano and likes to describe her as a "tough lady," said her determination to master the challenges of the institution is unyielding. "She is always on the job," Brennan said. "I don't always agree with her, but I'm always glad that she is on the team."
It is noteworthy that those disagreements rarely surface publicly. Few on the outside, for example, know that she was an early opponent of setting a one-year deadline to close the Guantanamo Bay prison facility. She has disagreed with Attorney General Eric Holder and Secretary of State Hillary Clinton on other matters, typically drawing a hard national security line.
But she seems uncomfortable having her views known. Asked about her opposition to the Guantanamo deadline, Napolitano grew quiet, then asked: "Who told you that?"
After a moment, she tried a diplomatic reply. "It became evident pretty quickly . . . that the deadline would have to be extended," she said speaking for several minutes, never fully confirming her disagreement with the original policy. But never really denying it.Planning her leisure
Before heading to Barcelona and Nigeria, Napolitano stopped in Madrid for a security conference, spending hours in long meetings. That night, she put into practice one of her life maxims: "Plan your leisure." That sounds like a killjoy, but without it, enjoyment would be even more rare.
And so, late on a Friday night, her entourage set out from the hotel to a restaurant. It was a conspicuous movement: Bulky men in dark suits cleared a path ahead, making sure it was safe for the compact woman in a red blazer to walk the streets of Madrid. Down a narrow alley, the secretary spotted a silk store and darted inside -- to shop for someone else, as usual. After thoroughly examining multiple scarves and summoning an aide for help, she settled on a blue one for her assistant of 11 years.
Some politicians have constant turnover on their staffs; others keep their aides for life, as Napolitano does. Maybe it's her gift-giving, or perhaps her offbeat sensibility: If Reagan loved jelly beans, and Pelosi is a chocoholic, Napolitano is avowed connoisseur of Peeps, the sugary Easter candy.
And then there's the singing. Her love of song is such that over the course of a single dinner in Madrid -- with wine flowing freely for everyone but the secretary, who quit drinking two years ago -- Napolitano serenaded her table no fewer than six times, her repertoire ranging from "American Pie" to the Girl Scouts anthem to the theme song from "Mamma Mia." She recalled once singing in front of Brennan, and said, laughing gregariously, that she was sure the serious terrorism adviser was horrified. "He was probably asking himself, 'Didn't they vet these people?' " she said.
In Barcelona the following day, she took another detour. After her port security visit, she headed to a Picasso museum, where she found a portrait that pleased her: "La Nana," the painting of an angry-looking female dwarf.
Napolitano laughed approvingly. Then she turned around, hands on hips, and assumed the role of the dwarf. "I'm tough," she declared. "Do not mess with me." Hours later, she was on her way to Nigeria, with the same message.


text:
What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
Can I do this with data from a form that has dynamic fields?Asked by poudreriver on December 14, 2016 at 04:03 PM
Can I do this with data from a form that has dynamic fields?
I want to create a registration form where any number of children's names are entered. If the family has one child, only one field will be filled in. If they have 3, they will be able to add 2 more. Is it possible then to pass the children's names to another form? I won't know how many fields there will be to send the data from.
This is a re-post of a comment on How to Automatically Pass Form Data to Another Form
JotForm Support
Hello, I checked the most recent form you edited https://form.jotform.com/63480619855163 and noticed that you already have something in place.
However, what you used for the fields is a widget, the Configurable List widget to be exact. If you aim to pass form data and prepopulate it to another form, it's best to use regular form fields (e.g. 1st Textbox for the First Name, 2nd Textbox for the Last Name, then 3rd DateTime field for the date).
You may then use the same logic for your Conditions to SHOW MULTIPLE fields based on the number selected on the Number of Students dropdown menu.
Afterwards, refer back to our guide on How-to-Automatically-Pass-Form-Data-to-Another-Form
I highly recommend you use our Prepopulate app http://prepopulate.jotform.io/ to get the fields needed easier.
As a side note, you have to keep an eye out with your daily API usage when using a lot of widgets. To learn more, here's a short guide on Daily-API-Call-Limits.
Thank you so much for your help. :)


text:
SpaceOut FREE
SpaceOut is a game where elements of breakout and space invaders come together to create an exciting arcade action experience! • Take control of the newly developed Plasma Tank and defend the earth from the incoming hordes of Arkanoids! Use your plasma balls to knock them out of the sky, all the while avoiding their bullets and collecting their de…
Free • 240


text:
I'm not asking whether or not I'm cute-- that's your opinion, I'm sure plenty of people think I'm ugly and some disagree but that's not what I'm asking, the internet is a brutal place for that, lol. I'm just looking for beauty tips, maybe things I can change (aside from plastic surgery or something drastic) to look the best that I can look. It's always good to get other people's advice! So here are two pictures, let me know some good suggestions. I'm looking to change things up a little bit. Even though I'm nowhere near as cute, I'd like to go for Victorias Secret "style" looks, I just like that style.
(in this one I'm all the way to the left)
Thanks guys! Help from stylists or professionals is even more appreciated :)
Most Helpful Guy
You might go with some choices in necklaces. That would really help with both the pictures provided. Try looking at Tiffany's Jewelry for ideas and try to find those picks cheaper somewhere else. You are very attractive.00|00|0
Thanks, I almost never wear necklaces. I don't know why. Tiffany's is way too pricey for me but I think I'll take a look at some nice drapey stuff in random stores.
You know I was thinking that with those turquoise ear rings you could get away with a leather lace with beads. And, also have you seen the necklace that Jamie Lee Curtis wore in Freakie Friday. It is a bit less conggestive than the draping neclaces.


text:
What if when the straw hats reach the Wano Country or any other island famous for swordsmanship one of zoros swords had broken in battle and a famous sword maker who made many of the Saijo O Wazamono grade swords makes a sword for zoro with all the physical features of the ultimate named sword (meito) and with zoros skills it makes the name of the sword spread throughout the one piece world and the sword becomes knowon as the thirteenth Saijo O Wazomono grade sword. I would love to see the expression on tashigi's face if this happens i mean zoro would have shusui, the wado ichimonji ( kitetsu would be the broken one) and this awesome new sword. Even Mihawk would probably be surprised and also many sword hunters would target zoro making it a more fun adventure.
what do you guys think is it possible or just a pipe dream???