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Surviving and Thriving During the Mercury Retrograde
As I scroll through my social media I start to see the warning of the cosmic frustration that we are about to endure — Mercury is about to go retrograde (cue the panicked preparation). Now, take a deep breath in, because what I'm about to tell you might shock you — this time can actually serve you and work for you, if you just let it. Okay, exhale. Let me explain the basics and benefits of Mercury's travels.
Mercury is the messenger of the gods, ruling over Gemini and Virgo, the two best communicators in the zodiac. In fact, the Roman god Mercury is directly associated with Hermes, the Greek messenger god. Mercury aids in enhancing curiosity, logic, reasoning, creativity, and thought processes. When Mercury goes retrograde, the planet moves differently. It gives an illusion that it is moving backward, and when the planet shifts, the energy of the planet shifts. Since the illusion of it moving backwards is just our perspective, so is the illusion that this cosmic time is negative.
Since Mercury aids in the magic of communication, the way we think, speak, and interpret things gets easily confused. Electronics seem to break, brain fog can cloud you, and little tiffs and inconveniences can happen in relationships and at work. It is a time when everything seems to go wrong. It is by far the most famous planetary time, and for good reason! When we can't communicate or process our thoughts, feelings, and emotions properly, things are bound to go wonky.
However, this is opportunity in disguise. You can harness this energy to get really clear on what is for you, and what you should shed and leave behind. Mercury retrograde seems to do the heavy lifting for us, it breaks off whatever is in the way of your path. Take some space for yourself and go inward. Notice what comes up for you. Maybe your travel plans went awry, or your hard drive wiped out, or there was a massive miscommunication at work. Take space and reflect on these situations. The trick here is to go with this divine flow. Mercury retrograde gives an opportunity to test your faith, get really clear about what you want, and puts you in situations where you can see new perspectives and gain new magic.
During this time, wait to start new endeavors and think about things carefully. Reflect on things you have set into motion or prioritize unfinished business. It's an ideal time to analyze what you may have overlooked or want to do differently.
This is a time where you can feel overwhelmed or anxious, since Mercury rules the nervous system. This doesn't help with the already strained communication. You may also notice people from your past coming out of the woodwork, which can lead to old feelings coming up. This is an opportunity to heal things from the past, practice being present and grounding in the now, and process all that is happening in your life.
Let your mantra during Mercury retrograde be "flow over friction." If you feel like you are holding on to ideas, outcomes, relationships, contracts, and things of the like too strongly, let it go and see how Mercury is helping you out. If you are noticing friction come up for you, that is Mercury holding your hand and guiding you to your truth. Take a step back and look — this is a gift.
Mercury retrograde is a time to own yourself. You have nothing to worry about, simply honor your thoughts, words, and deeds, and have them come from a place of integrity and love. So, breathe deeply and don't buy into the drama around this time. Let it be a helping hand to being fully you.
The intense dread we feel when we hear a planet is about to turn its energy inward, is a direct reflection of how we see the art of surrendering. These reactions highlight our wounding. Retrogrades are very yin, they change up the energy to an inward experience. In a world where all we want to do is produce, can we do as the cosmos do and take a necessary turn inward? We are made of stardust, after all.


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Today is Valentine's Day, the day the whole world seems to go crazy with displays of "love". Everywhere you look there are flowers, chocolate, and public displays of affections. It reminded me of a piece from my book about legacy, All I Have Needed-A Legacy for Life. Today I am posting that piece. You see, I didn't learn about love from society, television, or silly cards. I learned from people who knew what love was really about. So, here you are. Enjoy!
Public Displays of Affection . . . or Not
My dad's parents were ranch people. They grew up in Nebraska during simpler times when the work was hard and there weren't many frills. They lived in a sod house (where Daddy was born), and they lost everything during the Depression. When people talked about the "good old days," Grandma would say, "They weren't so good." They were loving but not demonstrative, at least not toward each other in front of others. That wasn't their way.
When Grandpa was about ninety, he developed a lump on his neck. He ignored it as it got bigger and bigger. After all, he was ninety. He didn't expect to live forever. One day it started causing trouble with his breathing, so they took him to the hospital, rushed him sixty-five miles from Broken Bow to Kearney, Nebraska. That lump had to be removed.
The morning of surgery, the staff came in to take Grandpa to the procedure and told Grandma, "You can kiss him goodbye if you want." To my parents' amazement and delight, she did. It wouldn't be considered a romantic moment by today's standards, but it certainly impressed Daddy. After all, at age sixty-five he was watching—for the first time—his parents kiss.
During the preparations for surgery, Grandpa's IV came apart, and he bled some. He bled enough that the doctors decided they should take him back to his room and check his heart before doing surgery. After all, he was ninety. Once he was cleared for surgery, Grandma had her chance again, and she went for it.
"Twice," Daddy said. "I saw them kiss twice!" The look on his face when he was telling us was priceless. It was pure delight and comfort. Proof of what we all knew. They loved. (As if one hundred direct descendants and sixty-five years of marriage wasn't enough proof.)
A couple of years later, Grandpa was hospitalized with a mild heart attack. It was caused, it turned out, by prostate cancer, and he was dying. Grandma, herself well into her 80s, couldn't care for him at home, so they put him in the nursing home attached to the hospital. During the next six weeks, Grandma went up every day to eat lunch with him … well, at least until she figured out that he wasn't eating when she was there in hopes she would take pity on him and take him home. She certainly wanted him home, but it was impossible, so she started going after lunch and sitting with him all afternoon.
Grandpa kept asking to see my youngest niece, who was about six months old, because he hadn't seen her yet. So, my sister and I took the children and drove down to visit for a few days. Grandpa was miserable and in pain, but uncomplaining.
One afternoon, Cheryl brought Grandma to the nursing home to visit. We pushed her wheelchair up close to the bed where they sat in silence, holding hands. He peered at her and asked, "Do you have enough money at the house for food?"
She smiled and assured him, "Yes, George, I have plenty." I understood something that day. Love isn't just about hugs and kisses. It isn't about telling someone they are beautiful or handsome. It certainly isn't just about romance. Love is about caring and providing. It is about sharing your lives, both the good and the bad.
I wonder, in today's world, where demonstrations of affection are everywhere you look, is it really better? After all, Grandpa and Grandma Jones were married for sixty-seven years. Theirs was a grand love story, and no one who knew them would doubt it for one moment. I sure didn't doubt it that day in the nursing home.
"When we were young, there wasn't much divorce. If you didn't like each other one day, you just waited a few days until you did." ~ Grandma Jones


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Farmhouse Golden Smoked Oak Brushed & Lacquered Engineered Wood Flooring
Farmhouse Golden Smoked Oak is the quintessential rustic floor that takes your breath away at first sight with its gorgeous organic details and textures. The warmth of the colour palette, with delicious tones of caramel, toffee, honey and russet accents can give any room a welcoming look and feel, creating a peaceful haven for you and your family.
The rustic-grade grain, knots, splits that have been carefully filled, and unique marks, all of these elements ensure Farmhouse Golden Smoked Oak looks and feels incredible from any angle. Completed with a 6mm American oak layer and brushed and lacquered to give its surface a high quality finish, this is a floor you'll be able to admire for a lifetime.
The rustic-grade grain, knots, splits that have been carefully filled, and unique marks, all of these elements ensure Farmhouse Golden Smoked Oak looks and feels incredible from any angle. Completed with a 6mm American oak layer and brushed and lacquered to give its surface a high quality finish, this is a floor you'll be able to admire for a lifetime.
A Closer Look
Farmhouse Golden Smoked Oak
20MM THICKNESSWith a hard wearing 6mm real oak top layer, this impressive 20mm engineered wood floor is ready for the hustle and bustle of family homes.
AUTHENTIC WOOD KNOTSThe random and natural appearance of knots and colour variations are central to creating the authentic wood feeling, showing off the character and class of the wood veneer.
HIGH QUALITY TREFFERT LACQUERA lacquered finish adds additional strength and protection to the hardwood whilst maintaining it's natural character and charm. Treffert are renowned as one of the world's most innovative leaders in the flooring finishes industry - they provide specialised coatings that protect the beauty and class of the flooring. This Treffert coating will keep your flooring looking great much longer than products coated with inferior coatings.
FIXED PLANK LENGTHSFarmhouse Golden Smoked Oak has long and wide fixed length planks, giving a neat and uniform finish with very few joins. Some packs will include starter boards, which will be 500mm in length.
SIDED BEVELThis engineered flooring is finished elegantly with a soft bevel rather than a harsh v-groove, giving subtle class to the floor rather than the slightly more unnatural v-groove. The 2 sided bevel additionally gives the appearance of longer plank lengths
GRAIN VARIATIONAs a natural product wood will bring a variation in the look and character of the flooring, the amount and size of the grain, knots and growth rings all add to the uniqueness of wood flooring.
NATURAL GRADE FRENCH OAKFrench Oak is a high quality, natural grade product that guarantees character and authenticity. Sourced from only well maintained, sustainable forests, French Oak is full of natural variety.
NATURAL WOODOur woods are a natural product and as such can have naturally occurring imperfections. These imperfections are remedied by filling with a hard resin which is materially stronger than the wood itself.
SUSTAINABLY SOURCED FLOORINGThis flooring has been sourced from an approved source, meaning that it has come from only the most well maintained and sustainable forests. This helps preserve delicate ecosystems and ensures that native cultures and economies are respected.
CHARACTER GRADEOur Character Grade sits in between our Rustic Grade and Prime Grades. It doesn't have as much colour variation as our Rustic Grade but still includes knots and cracks to give it a unique style. If you are looking for a floor with little colour variation and few knots or cracks then we would recommend looking at our Prime Grade woods.
Our Platinum warranty covers this product for life from manufacturing defects to do with click system and also the tongue & groove mechanism.
For more information, read our warranty page.
INCREDIBLY HIGH QUALITY PLYWOODOur plywood is a very high quality plywood sourced from managed forests in South East Asia.
Some products are known to have a low grade Chinese plywood backing, faced with Poplar (which is a hardwood) but with a softwood core and still be using the same description.
Suitable for underfloor heatingAll our engineered wood is suitable to be laid over underfloor heating.
Farmhouse Golden Smoked Oak
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20mm Thickness
6mm Hardwood Layer
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Director Lingusamy is planning to remake Vettai in Hindi. The director is looking to strike gold in Bollywood and feels that Vettai would be his ticket. Talks are on with Salman Khan to essay the role Arya did in Vettai. It is also believed that Amala Paul may get a chance in the Hindi version as well. The stars for the roles played by Sameera Reddy and Madhavan are yet to be finalized, say sources.
Vettai is slated for release this Pongal. Music for this film has been scored by Yuvan Shankar Raja. Arya, Amala Paul, Madhavan and Sameera Reddy play the lead roles in this film.


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Wedbush Morgan analyst Michael Pachter is out with another prediction, claiming eight million motion controlling devices, both the PlayStation Move and Kinect, will be in homes by the end of 2010 and over 20 million by 2011.
"Given recent guidance from Microsoft, it is likely that the two peripherals will have an installed base of 8 million by year-end, and depending upon pricing, we could see an installed base that approaches 20""25 million by year-end 2011," he said.
"These 20""25 million households are likely to buy 2""3 games apiece, resulting in software sales of 40""75 million units and publisher revenues of $1.6""3 billion next year. These sales are not all incremental; clearly, consumers who buy Kinect or Move are console owners, and presumably, they would have bought other games had they not purchased the peripherals.
"We think that the incremental contribution from the two devices will be around $500""800 million in 2011, or around 2.5""4 percent of industry sales next year."
Sony's managed to move 4.1 million PlayStation Moves worldwide while Microsoft has shifted 2.5 million Kinects so far with the current goal of 5 million by the end of the year.
Source: VG247


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To help ensure that your wishes are clearly communicated, Neighborhood Christian Legal Clinic is offering a new program to assist individuals with their Wills and Estate needs. Estate Planning services include Simple Will, Power of Attorney, Designation of Healthcare Representative, Quitclaim Deed, After Death Transfer Documents, and more. Initial consultations are free of charge, while all other services are available according to a discounted flat-fee schedule. This program is meant to serve those with moderate to low incomes.


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In accordance to a report by the BBC, Fb has severe plans introduce its personal crypto foreign money. The fb coin, internally referred to as 'GlobalCoin' will be introduced in a number of international locations in the primary quarter of 2020, after in depth testing on the finish of this yr.
The brand new stablecoin which has been rumored about for a number of months now, will allegedly be used for Facebook's personal fee system in which customers will be in a position to switch cash. The system might give Fb useful insights into how, and on what, customers spend their cash.
The objective behind it's easy, If the fee system hits off, it might quickly compete with banks and performance as a world, on-line system with dramatically diminished charges. Fb needs to create a digital foreign money that gives inexpensive and safe methods of creating funds, no matter whether or not customers have a checking account.
In accordance to the BBC, Fb is probably going to hyperlink its cryptocurrency to fiat cash (fiduciary cash), such because the euro, greenback or yen, to stop a fluctuating change fee with massive will increase or decreases in worth.
Fb has reportedly been talking to numerous corporations and authorities businesses to make certain the regulatory aspect is permitted. The corporate apparently has already spoken to Financial institution of England governor Mark Carney, a dialog in which they mentioned the alternatives and dangers concerned in launching a cryptocurrency. Fb appears to have sought recommendation on operational and regulatory points from officers on the US Treasury as properly.
For the industrial aspect of issues, the agency has been speaking to Western Union in a quest to search for cheaper and sooner methods for individuals with out a checking account to ship and obtain cash.
The BBC report additionally mentions that there are some considerations concerning the new fee system. Lately, Fb has come beneath fireplace over its dealing with of customers' private knowledge, and regulators are doubtless to study the launch intently.
Earlier this month, the US Senate and Banking committee wrote an open letter to Mr Zuckerberg questioning how the foreign money will work, what shopper safety will be supplied and the way knowledge will be secured.
In accordance to BBC, Fb has not responded to the report of the British broadcaster.
- Beneficiant Donation of $120,000 in Bitcoin by Nameless Donor Very Nicely Acquired by Bitcoin.org – Might 30, 2019
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- Bitbond, a International Lending Platform, Launches STO to Assist Small Companies in Asia with Loans – Might 28, 2019
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- Visa Token Service Acquired by Eleven New Companions for Enhanced Shopper Confidence in the E-Commerce House – Might 23, 2019
- Voyager Pronounces Integration of Ledger Vault's Asset Administration System into its Crypto Buying and selling Platform – Might 22, 2019
- VeganNation Welcomes Recreation of Thrones Actor Jerome Flynn to its Advisory Board to Unite the Vegan Group – Might 21, 2019
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- Q2 Holdings Pronounces the Launch of Q2 TrustView, a brand new Governance and Safety Expertise for Banking and Lending – Might 15, 2019
- First Safety Token Blockchain known as Polymesh introduced by Polymath at Consensus New York 2019 – Might 14, 2019
- Fb lifts Crypto Advert Ban, One Yr after the Social Media Big Banned all Advertisements Associated to Blockchain and Cryptocurrencies – Might 9, 2019
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- With Hoard, Video Recreation Builders Will Now Have the Skill to Seamlessly Combine Video games with Ethereum – Might 1, 2019
- ShelterZoom Creates Contract and Transaction Administration Platform Marking New Contract Evolution on Blockchain – April 19, 2019
- Samsung SDS and Tech Mahindra Be a part of to Discover International Blockchain Market utilizing Samsung's Nexledger Platform – April 15, 2019
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The world market leader in the field of windsurf for over 20 years, BIC Sport has diversified into the manufacture of surf boards, kayaks, sailing dinghies and Stand Up Paddle boards. This latest brand new board sport is largely based on the principles of simplicity and accessibility that have underpinned BIC's enduring success.
BIC Sport has become the world's leading expert for water sports. A forerunner in the industrial scale manufacture of surf and SUP boards, a benchmark manufacturer in the demanding surf market, BIC Sport is also a European market leader in sit-on-top kayaks, with a range of innovative kayaks that have picked up numerous design awards.


text: WiszdomStone Rise by Michael Popke Mar. 22, 2010 midnight Back to Search Results × Expand × Rise is aprogressive-metal tour de force that comes along at the right time, as a newgeneration of headbangers raised on Dream Theater, Symphony X, Kamelot, TadMorose and Seven Witches seeks out similar bands. This is WiszdomStone's firstfull-length album, and despite slightly muddied production and a fewby-the-numbers duds, rousing songs like "Unforgiven," "Take This Cup" and "TheSky Is Falling" should convince a label such as Nightmare Records or NuclearBlast to reissue this independent release to a wider audience. What's more,guest appearances by former Queensryche guitarist Mike Stone and Kamelotdrummer Casey Grillo already give WiszdomStone street cred.


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LEDTOUCH™ Cylinder Downlight series is the ideal energy-saving replacement for 35W, 70W and 150W metal halide downlights, which provides high quality lighting for high-ceiling spaces for both indoor and outdoor applications. Intelligent control is available on request for better energy efficiency and operation efficiency.
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In this episode of the Doom and Bloom Survival Medicine Hour with Joe Alton, MD and Amy Alton, ARNP, a Florida landfall occurs for a hurricane for the first time in more than a decade. Were you ready? What should you do to prepare for the next one? Plus, Dr. Bones discusses new tragedies for the native bee population in the U.S. What will be the straw that break's the bee's, I mean, camel's back? With every third bite of food you put in your mouth coming as a result of some bee pollinating a plant, you should be invested in this topic!
Plus, we haven't talked about Zika for a while, but that's not because there hasn't been a lot of news about it. Nurse Amy and Dr. Bones follow the globetrotting pandemic to a new outbreak in Singapore, talk about outbreaks that might not be reported due to lack of testing, and the effects that could occur on zika-infected newborns that are born looking perfectly normal.
All this and more on the latest Survival Medicine Hour with Dr. Bones and Nurse Amy!


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Santo Tome del Puerto
Venta Juanilla Santo Tome del Puerto
Venta Juanilla Santo Tome del Puerto
Ctra. N-I · Madrid-Irún, Km. 99
Santo Tome del Puerto Spain
Rated 79 / 100 based on 37 customer reviews.
The quality of service and hospitality at Venta Juanilla Santo Tome del Puerto has been identified as outstanding based on consistently high customer review ratings.
The customer review score on Gohotels is calculated using a proprietary algorithm to determine an overall customer rating between 0 and 100. Multiple factors such as cleanliness, facilities, location and service are considered.
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Venta Juanilla Santo Tome del Puerto has achieved a score of 79 / 100 based on 37 customer reviews and has therefore qualified to receive an Outstanding Service Award from Go Hotels.
If you own or represent this business click click
to learn more about this award and how it can benefit your business.
Set in a 10th-century building with a stone facade, this relaxed hotel is 10 km from the Chorrera de los Litueros waterfall and 11 km from the Cueva de los Enebralejos (cave).
Offering garden and/or mountain views, the understated rooms come with free Wi-Fi and flat-screen TVs. Suites add separate living and dining rooms.
Parking is free. There's also a laid-back restaurant featuring wood-beamed ceilings and a casual bar.
Facilities @ Venta Juanilla Santo Tome del Puerto include:
Business Center
Tour Desk
Elevator / Lift
24-Hour Reception
Air Conditioned
Multilingual Staff
Safe-Deposit Box
Meeting Rooms
Games Available
Coffee Shop / Cafeteria
Luggage Storage
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text: bonsey 1 Report post Posted December 30, 2017 Hi Folks, new user here looking for some help. Just got a Vinyl Express R31 from Signwarehouse. Having trouble getting it to cut from the VE LXi Expert 12 software. I installed the cutter via USB on Windows 7 32bit with the drivers that were listed in the instructions. The computer sees the port. I configure the Lxi Production Manager with the cutter settings and the correct port #. I can do a "Test" from Production Manager and it cuts a small bullseye shape. If I open LXi Expert and try to do simple "TEST" cut, the cutter goes all the way to the left and the online blinks red and it has to be power cycled. It does not actually make the cut. Also under the Devices and Printers section in Windows, it shows as "Printer for COM11" and in the properties it shows "Model: Send to Microsoft OneNote 15 Driver". So when the cut is sent from LXi Expert, it also trys to open Microsoft OneNote. Not sure where to go from here. Seems there is a disconnect between LXi Expert and the Production Manager or driver issue with Windows? Any tips or suggestions would be great. Thanks for your time ! Share this post Link to post Share on other sites


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Category Archives: Dolliecholia
Alex gets new clothes
New clothes from SartoriaJ. Advertisements
Posted in Dolliecholia Tagged ball jointed dolls, BJD, dollshe Leave a comment
Varla's Wounds
Here's the map of how love hurts. Alex took her sweet time making Varla hers: 23 cigarette burns two knife cuts (one near her breast and one in her side) one brand of a scarlet A on her right breast … Continue reading
Posted in Dolliecholia, Uncategorized Tagged ball jointed doll, BJD, fairyland minifee, Mirwen Leave a comment
Of Women and Bondage
Got my harness today from SyntheticEphemera and Alex has been looking forward to this (well okay, me too). It's an early Valentine's Day present. It's cool though fussy to get on. I could have used an Ikea diagram. Hmmmm…..I like that … Continue reading
Posted in Dolliecholia Tagged abjd, ausley, ball jointed doll, BJD, dollshe, dollshe ausley Leave a comment
Hangin' out watchin' movies
I had to watch Shoah, The Sorrow and the Pity and Night and Fog for my film class this week (The 27th is International Holocaust Remembrance Day) so I signed up for SundanceNow Doc Club and Alex and I sat … Continue reading
Posted in Dolliecholia, Uncategorized Tagged dollshe, dollshe ausley Leave a comment
The Love Story of Alex and Varla
Varla is my Minifee Mirwen, yet to be painted. She is Alex's lover (yes breaking DoA protocol with SD's and MSD's having intimate relations.) I want Varla to be dark. She's got scars as you're about to read. Lots of … Continue reading
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Don't Fear the Reaper
Nibelung, riding a skull dreaming of the day his master, Gilles De Rais, comes. He knows that someday soon the curtains will flew and then he'll appear. When that happens he knows that his appetite for destruction will find it's … Continue reading
Posted in Dolliecholia Tagged abjd, ball jointed doll, BJD, death, doll chateau, doll-chateau ada, skull Leave a comment
Just Hangin' Around
Celine and Cassandra listening to The Stranglers and letting the night flow by….
Posted in Dolliecholia Tagged abjd, BJD, Dream of Doll, Dream of Doll Tahlia, Peak's Woods Wake up Lottie, Peaks Woods Leave a comment


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There are a number of reasons which are responsible for foot pain, arthritis can be one of them. If you are suffering from arthritis then joint and foot pain are common problems. Any person can become a victim of foot pain.
With the advancement in technology updated procedures are invented for arthritis and joint pain treatment. Nowadays PRP therapy is getting popular. There are a number of people who are taking stem cell therapy for foot and joint pain. You can browse this website – https://medicastemcells.com/ if you want to know about stem cell therapy.
Brief information about Platelet Rich Plasma therapy:
In this treatment patient's blood is used for the treatment of ankle pain. Blood is taken from the body of the patient from the region which is rich in platelets.
In order to separate platelets from the blood, it is spun with a centrifuge. This process separates red blood cell, plasma, and white blood cells. The platelet-rich plasma is injected directly to the affected area of foot and ankle pain.
This therapy has no side effect as a patient's own blood is used. This is the preferred advantage of this treatment.
How PRP therapy heal foot pain?
As already explained about the reason for foot pain can be torn ligaments or arthritis. So PRP therapy is great to heal the foot and ankle injuries.
- Platelet-rich plasma which is injected in the affected area helps in healing the torn ligaments and joints. This recovers the torn tissues which lead to healing of foot pain.
- This plasma increases the production of collagen in the affected area which further enhances the healing process.
- This is a noninvasive treatment to heal the torn ligaments of foot and ankle with no side effects. This therapy also has fewer post-treatment issues as compared to surgical procedures.
You can discover more here about the platelet-rich plasma therapy for foot pain. You can find online experts who are providing the services of PRP therapy.


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Former Self stars congratulate coach on 600th victory
In case you haven't seen it, here's that video that played last night with 15 former Bill Self players (including 11 Jayhawks) congratulating him on win No. 600.
Familiar faces (near and far) offering congratulations to @CoachBillSelf on his monumental 600th career win! #kubball pic.twitter.com/xZjoxQxSOh— Kansas Basketball (@KUHoops) December 7, 2016
Frank Mason's game winner vs. Duke, up close and personal
If you're like most Kansas basketball fans today, you just cannot get enough of last night's victory over No. 1 Duke.
Whether that means you've been surfing the web to read as much as you can about the 77-75 victory — thanks, by the way! — or you've been on Twitter, Instagram, Facebook and anywhere else you can think of to view imagines of Frank Mason's thrilling game-winning shot, the aftermath of that moment or any other images of the game, your day likely has included you smiling a little bigger, puffing your chest out a little farther and perhaps even reaching out to those Duke fans you know (and sometimes even love) to tell them "good game" or some other version of that thought.
But if you still haven't got enough of the shot — and what a great, clutch shot it was — take a look at this video, filmed at courtside, of the final moments of Tuesday's game and Mason's terrific game winner.
It's no doubt the biggest shot of Mason's career to date and it came on the biggest and brightest stage college basketball possibly can have in mid-November.
One of the great things about Mason being the one who hit the shot is that the Jayhawks run absolutely no risk of the moment going to his head. Mason on Friday in the home opener against Siena is going to be the same player he was on Monday morning, Tuesday at halftime or when Duke hit the three-pointer to tie the game — an ultra-competitive, fighter who is willing to do anything necessary to help his team win.
If that means he takes the shot, he'll take it. If that means passing the ball, playing D or finding a way to make a big steal, you can bet Mason is going to do whatever he can to get the job done. It doesn't mean he's always going to succeed, but few Jayhawks in recent memory have been as willing to get dirty and lay it on the line like Mason and that's what makes Tuesday's game winner such a big deal. Because of it, Mason is finally getting his due on the national scene.
Enough about all of that, though, let's get you to the awesome video of the big shot.
Special props to those of you who watch it enough times to catch a few glimpses of KUsports.com photographer Nick Krug right there in the thick of the celebration.
Looking back at Late Nights past
With the 32nd annual Late Night in the Phog a little more than 24 hours away, the anticipation of the beginning of another Kansas basketball season is at an all-time high.
Led by 14th-year head coach Bill Self, the Jayhawks will open the season, as they always do, with high hopes and lofty goals. A likely Top 3 team heading into the season — which officially begins in Honolulu on Nov. 11 — the Jayhawks will be gunning both for a national title and a record-tying 13th consecutive Big 12 regular season title.
While the quest for both will hold the interest — and nerves — of KU fans for the next several months, few things get the fan base as fired up as Late Night, which offers both an opportunity to see the players in action and be entertained by their personas away from basketball.
"There's nothing like it," said junior guard Devonte' Graham. "The fans know what recruits are coming from high school. They get all the privileges just to be around here and experience Late Night. Seeing Allen packed is different from just walking in and seeing it empty. You can't really imagine it but it definitely is a huge impact on recruits."
Added Graham backcourt mate Frank Mason III: "I'm looking forward to having a great time with my teammates for the last time. I think it is good for freshmen to play in front of the crowd. It's a great thing we do every year and I'm looking forward to it. The skits are always funny. I'm not used to dancing and things like that. I think the dance moves are hilarious and us trying to fit in with the dancers. It's always a good time."
With that in mind, here's a quick look back at recent Late Nights, through the lens of our talented photo staff...


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AARP Virginia State Director will be part of the Virginia College Affordability Policy Council Read More »
Join us for lunch or dinner in October! Have fun eating and socializing with members and friends of the AARP northern… Read More »
Remember pouring over the "Help Wanted" section of the classified ads in the newspaper when looking for a job? Remember… Read More »
AARP Smart DriverTEK Workshop Features New Safety Technologies Read More »
AARP Community Ambassador Olivia Dabney has been volunteering with AARP for two years but has been serving her Virginia… Read More »
Deadline to Apply for Mailed Absentee Applications is October 29 Read More »
Join AARP Virginia at one of our many local events Read More »
Exploring new interests and finding the time to pursue old ones, getting legal documents in order, decluttering the… Read More »
Saturday, Sep 21, 2019 at 11:00am
Arlington Public Library
Arlington, VA
Sunday, Sep 22, 2019 at 12:00pm
Springfield Town Center
Springfield, VA
Monday, Sep 23, 2019 at 10:00am
Arlington Central Library
Arlington, VA


text:
Multiple attributes of the three-dimensional (3D) extracellular matrix (ECM) have been independently implicated as regulators of cell motility, including pore size, crosslink density, structural organization, and stiffness. However, these parameters cannot be independently varied within a complex 3D ECM protein network. We present an integrated, quantitative study of these parameters across a broad range of complex matrix configurations using self-assembling 3D collagen and show how each parameter relates to the others and to cell motility. Increasing collagen density resulted in a decrease and then an increase in both pore size and fiber alignment, which both correlated significantly with cell motility but not bulk matrix stiffness within the range tested. However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment. Cellular protrusion rate, protrusion orientation, speed of migration, and invasion distance showed coupled biphasic responses to increasing collagen density not predicted by 2D models or by stiffness, but instead by fiber alignment. The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified. Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.
Cell motility through 3D extracellular matrix (ECM) is a key biological process involved in normal development and homeostasis, as well as the progression of diseases such as metastatic cancer. Efforts to understand the invasion and movement of cancer cells through the collagenous ECM surrounding tumors, a key step in metastatic progression, originally used 2D model systems that allow the complexities of the microenvironment to be significantly decomposed. Now, however, multiple studies have highlighted the major differences between 2D and 3D cancer cell motility1,2,3,4, and 3D systems have become standard. Within these 3D systems, several physical features of the extracellular matrix (ECM), i.e. stiffness5,6,7,8, ligand density9,10,11, crosslinking6, and pore size12,13, and fiber alignment14,15,16 have been implicated independently as driving factors of cancer cell motility. Most studies have focused on only one or two matrix parameters, despite the fact that a change to any one parameter almost always affects another, or they have used non-native polymers or digested ECM proteins that do not crosslink and form microstructures that are physiologically relevant17. An integrated understanding of how density (which is the most commonly used descriptor), ligand presentation, crosslinking, and microstructural organization are related to each other and to cell behavior is still lacking in the context of the native acid extracted collagen-based 3D ECM now used by many researchers.
Here we take an integrative approach to characterizing and understanding these convolved features by embracing the complex combinations of matrix parameters that arise naturally in 3D self-assembling collagen I networks. By creating collagen gels of increasing density over a six-fold rage, we generated multiple complex matrix features. Embedded cells were assessed for their motility behavior (cell speed, invasion distance, and protrusion dynamics) while the matrix itself was characterized for its physical features (stiffness, density, pore size, and alignment of fibers). Then additional enzymatic crosslinking achieved changes in matrix parameters independently of density changes. Cross correlations among these measurements allowed us to uncover a distinct relationship between fiber alignment and cell motility independent of pore size and bulk matrix stiffness. Central to our approach is the fact that in 3D collagen, cancer cells move into the 3D matrix, rarely retracing the void tracks they leave behind, and so are always interacting with consistent microstructural properties1,18 (see also Results section).
3D cell motility is biphasic with increasing collagen density
We first asked what differences in cell motility where characteristic of increases in ligand density in 3D collagen. Cell motility parameters, including speed, invasion distance, and number and orientation of protrusions of embedded HT-1080 human fibrosarcoma cells were systematically assessed as collagen I density was increased from 1-6 mg/ml. Interestingly, a biphasic dependence of multiple motility parameters with collagen I density was observed, which is opposite to what occurs for 2D cell motility with increasing ligand density9,19 and for what has been predicted for 3D matrices20. At low collagen I concentration in 3D (1 mg/ml), cells moved rapidly and persistently with a sustained high rate of protrusion formation, and invaded to distances far from their point of origin (Fig. 1A,F–H). Cells also maintained the orientation of their protrusions over 12 h (Fig. 1I,J), i.e. the large majority of cell protrusions remained polarized along the original axis of elongation of the cell. At intermediate collagen concentrations (2 and 2.5 mg/ml), in contrast to what would have been predicted from cells moving on a 2D substrate, cells migrated more slowly and invaded smaller distances from their point of origin than cells in 1 mg/ml matrices (Fig. 1 B,C,F,G). Cells also generated fewer protrusions (Fig. 1H) and the directionality of their protrusions was significantly more isotropic than cells in 1 mg/ml matrices (Fig. 1I,K,L). Finally, when cells were embedded in high-density collagen matrices (4 and 6 mg/ml), cell speed increased, but did not achieve speeds observed in 1 mg/ml matrices (Fig. 1D–G). Cells also increased their rate of protrusion formation (Fig. 1H) and became highly polarized once again (Fig. 1I,M), similarly to cells in 1 mg/ml matrices. MDA-MB-231 human breast cancer cells were used to test whether this motility response to increasing 3D collagen density was cell-type specific. Highly similar motility trends were observed for MDA-MB-231 cells (Supplementary Fig. 1A).
Cell migration speed, invasion distance, and the number of cellular protrusions were highly correlated with each other and with collagen density from 1–2.5 mg/ml (Fig. 1N–P). However, data for the higher concentrations of 4 and 6 mg/ml shifted away from this trend and were considered outliers (Supplementary Fig. 1D–F). To determine if properties of the matrix other than collagen density were involved in modulating cell motility and to understand the switch in behavior that occurred for 4–6 mg/ml densities, we next characterized the physical and architectural properties of each matrix.
Fiber alignment and pore size, but not matrix stiffness, correlate with cell motility
Since several physical properties of the matrix (pore size, stiffness, fibrilar structure, etc.) can change concurrently with changes in collagen density, we aimed to characterize these finer physical details and ask how these features varied with each other and with observed motility responses. Analysis of reflection confocal images of 1–6 mg/ml matrices (Fig. 2A) showed that fiber alignment (measured at length scales of the cell) varied biphasically with collagen concentration (Fig. 2B). The average pore size varied somewhat irregularly, but overall was reduced with increasing collagen concentration (Fig. 2C). These two matrix parameters correlated significantly with one another across all collagen densities (Fig. 2D). Quantitative shear rheometry was used to measure the bulk matrix elasticity, which varied somewhat irregularly with increasing collagen concentration (Fig. 2E). Although previous studies suggest that the elastic modulus should scale positively with increasing collagen concentration21,22, it is critical to note that the process by which collagen is extracted23, the gelation conditions22,24, and even the thickness of the matrix22 can all significantly alter its mechanical properties and related microstructure25. Collagen density alone, which is often the only information provided in studies, is not sufficiently descriptive to enable direct comparisons. In our collagen matrices, the alignment of fibers decreased significantly when collagen density was increased from 1 mg/ml to 1.5 mg/ml, but the average pore size did not change. That the mesh is not tighter, but is significantly less aligned likely explains the decrease in the elastic modulus from 1–1.5 mg/ml. Then from 1.5 to 2 mg/ml pore size decreases dramatically while fiber alignment drops only slightly. This means that 2 mg/ml represents a tighter mesh, which would be expected to increase the elastic modulus compared to 1.5 mg/ml. Comparing the measured changes in matrix microstructure and mechanics to changes in cell motility revealed a significant correlation of cell speed and invasion distance with both fiber alignment and pore size (Fig. 2F,G) across the varying densities of collagen. Cell speed and invasiveness did not correlate with matrix stiffness, at least within the range of collagen concentrations tested (Fig. 2H).
Additional crosslinking reinforces fiber alignment as a predictive parameter
Since local fiber alignment and pore size correlated with each other across a wide range of collagen densities, we developed a method to alter microstructure independently of density to try to decouple these features. This was accomplished by the addition of the crosslinking enzyme Transglutaminase II (TGII) to the matrices. Crosslinking of 1 mg/ml and 2 mg/ml collagen matrices by the addition of TGII allowed alteration of the microstructural properties of the matrices, while preserving global collagen concentration (Fig. 3A,B). Cells embedded in 1 mg/ml matrices further crosslinked with TGII migrated significantly more slowly than cells in non-TGII treated 1 mg/ml matrices (Fig. 3C). However, the migration of cells embedded in 2 mg/ml matrices further crosslinked with TGII was unchanged compared to non-TGII treated 2 mg/ml matrices (Fig. 3C). Addition of a ten times higher concentration of TGII did not further alter the ECM microstructure or cell speed at either concentration of collagen (Fig. 3C).
As a control to ensure that TGII did not affect cell physiology directly, we applied TGII to cells migrating on conventional 2D substrates at the same concentration as used to crosslink 3D matrices and monitored possible changes in cell behavior. Cell speed on conventional 2D substrates was not altered by the addition of TGII (Supplementary Fig. 1C), suggesting that our observed changes in 3D cell motility were specifically related to crosslinking of the collagen matrix and the ensuing alterations to its physical properties.
Our microstructural analysis of the matrices showed that collagen crosslinking by TGII reduced the alignment of the fibers in 1 mg/ml matrices (Supplementary Fig. 1B), but did not significantly alter the alignment of 2 mg/ml matrices (Fig. 3A,B(top inset)). These results fit the previously established correlation between cell speed, invasion distance, and fiber alignment (Fig. 3F) and further increased the significance of the correlation. However, average pore size was unchanged following the addition of crosslinking TGII for both 1 mg/ml and 2 mg/ml matrices (Fig. 3E,G), indicating that fiber alignment can regulate cell motility independently of pore size.
Requirement for MMPs critically depends on matrix microstructure
MMPs are thought to be critical for cancer cell migration and invasion in crosslinked matrices where the average pore size is significantly smaller than the cell body. Here cell size is an order of magnitude larger than the average pore size of the matrix, ~0.6–2.3 μm in diameter (Fig. 2C), so we wondered how the requirement for MMPs might be affected. MMP −1, −2, −7, −9, and the membrane tethered MT1-MMP, which are considered critical for cell migration and invasion26, were inhibited for cells embedded in either 1 mg/ml or 2.5 mg/ml collagen matrices using a range of Marimastat concentrations. The concentrations of 1 mg/ml and 2.5 mg/ml were selected for comparison because they represent the extremes of both cell motility and fiber alignment observed in this study (Figs 1 and 2). For 1 mg/ml, collagen I formed structures that were the most aligned and for which cells were the most invasive, protrusive, and uni-axially oriented (Fig. 1G,I,J and Fig. 2B). For 2.5 mg/ml, collagen I formed structures that were the least aligned, and for which cells were the least invasive and protrusive, and protrusions were more isotropically oriented (Fig. 1C,G–I,L and Fig. 2B).
Maximal inhibition with Marimastat at a concentration of 30 μM (higher concentrations resulted in cell death) did not abolish motility for cells in highly aligned 1-mg/ml matrices. Instead, they maintained ~50% of their invasive ability, on average 75μm over 16.5 h (Fig. 4A). Marimastat-treated cells in 2.5-mg/ml matrices (Fig. 4A, right inset) were essentially confined to the void in which they had been initially embedded. When MT1-MMP was overexpressed in cells embedded in 1-mg/ml and 2.5-mg/ml matrices, cell motility increased by an insignificant amount and 67%, respectively (Fig. 4B).
Taken together, these results suggest a threshold for MMP utility within a 1-mg/ml matrix for which collagen fibers form highly aligned microstructures. MT1-MMP overexpression did not alter the orientation of cellular protrusions in these matrix conditions (Fig. 4C, D). This suggests that regardless of whether more MT1-MMP mediated collagen degradation occurs, the directionality of motility remains unaltered and is driven and oriented by the fibrilar topography of the ECM.
We have conducted a multi-parametric quantitative analysis of cell motility in 3D collagen matrices of controlled density to understand how fibrilar matrix properties modulate motility. This study was designed to span a broad range and complexity of physical features of the matrix, including pore size, fiber alignment, stiffness, and extent of crosslinking, such that any key feature would be tested in combination with several features to avoid bias. After a comprehensive quantification of each parameter, we relied on cross-comparison of matrix parameters and motility measurements to identify the features of the matrix influencing cell motility. We find that the local alignment of collagen fibers is highly predictive of cell motility across the entire range and combinations of matrix conditions tested, while collagen density is highly predictive only at low concentrations. Pore size correlates with cell motility parameters, corroborating recently published findings13, but less significantly than fiber alignment. By incorporating a secondary method of crosslinking that modulated matrix architecture independently of density, the relationship between fiber alignment and motility was further reinforced while that of pore size, density, and motility was not.
Our collagen image analysis method provides a score for the pore size between fibers and the fiber alignment for fiber intensity signals that are greater than imaging background noise, which is removed during reflection confocal image processing (see Methods). Moreover, imaging resolution must also be able to resolve fiber locations spatially. In general, reflection imaging is limited in its ability to detect fibers that are oriented at an angle greater than ~50 degrees to the 2D imaging plane27 and by the spatial resolution of the imaging system (~0.20 μm/px in our case). If the average space between fibers is less than the spatial resolution of the imaging system, the analysis results can be unreliable. In Fig. 2A, fibers are distinguishable in all conditions, but the 6 mg/ml case shows significantly fewer distinguishable fibers. Additionally, the average pore size in 6 mg/ml approaches the imaging resolution limit (Fig. 2C), indicating a rationale for the larger variability in alignment measurement for the 6 mg/ml case (Fig. 2B) compared to all other conditions. Nonetheless, our method is able to make highly reproducible, quantitative comparisons among the other multiple collagen concentration and crosslinking conditions and to predict the effect of ECM architectural changes on cell motility with high statistical significance (Fig. 3F).
Our alignment analysis constitutes a bulk measurement for all the fibers in the imaging area (analyzed images of collagen cover 61 μm × 61 μm) and reports the degree of anisotropy of the image as a whole. Simulations show that our method for quantifying alignment is not sensitive to changes in the length of fibers, but is sensitive to significant changes in width (data not shown). However, the width of the fibers did not change significantly across the different concentrations of collagen.
The significant influence of fiber alignment, measured at a length scale that is of the same order of magnitude as cell invasion distance in our study, likely arises because of topographic guidance as the cell protrudes and attaches to the ECM. Indeed, we find that the orientation of cellular protrusions follows a similar trend to that of fiber alignment. During review of this manuscript, another paper appeared showing that aligned 3D collagen matrices enhance migrational persistence and orient cellular protrusions, corroborating our findings28. Other studies using synthetic nanofiber scaffolds have demonstrated that fiber alignment promotes cell elongation, restricts sites of cell attachment, aligns adhesions, and promotes faster migration speeds29. This imposition on cell shape has been shown to play an important role in traction generation on 2D substrates30, resisting repolarization on 1D substrates31, and in generating polarized traction forces in 3D, which are thought to facilitate invasion32. Using a native 3D collagen matrix, our studies identify fiber alignment as a key feature even in light of the introduction of many additional complex matrix changes.
In our study, matrix architecture is assessed without cells present in the matrix. Since cells continuously migrate into the matrix and do not typically turn to retrace the void space they leave behind (over our observation time of 16 h), and because we seed cells at a very low density, we anticipated that cells would continuously encounter the same conditions as dictated by the initial matrix architecture. That these initial matrix conditions predict cell motility indicates that this assumption is justified. The ability of cells to pull on and align the matrix after their initial adhesion to the ECM may also play a role, possibly feeding back to positively reinforce cell polarization. However, others have shown that cell invasion in 3D matrices correlates more strongly with cell shape elongation than with contractility32.
It is well established that increasing ligand density on planar 2D substrates induces a biphasic increase and then decrease in cell speed9. In 3D matrices, we observe the opposite: a decrease then an increase in cell speed. The 2D phenomenon is thought to reflect changes in adhesion strength mediated by the aggregation of proteins at focal-adhesion sites33. At high planar ligand density, strong adhesion inhibits cell movement, while at low planar ligand density, weak adhesion strength causes less efficient motility. Here, the fibrilar topology of the 3D matrices presents a distinct network of nanoscale, linearly organized ligands to which the cell adheres. A recent modeling study addressed this point and predicted differences in cell speed and invasion distance based solely on fibrilar matrix organization, finding a significant role for alignment34. Our study shows experimentally that the alignment of discrete ligand-containing fibers directs cell protrusions and motility distinctly in 3D35,36,37.
By treating cells in 3D matrices with Marimastat, a wide-spectrum MMP inhibitor, our studies show a near complete loss of motility for cells in matrices of intermediate density (2.5 mg/ml), where pore size averages 0.9 μm in diameter and where fibers and cell protrusions are isotropic. However, treating cells in low-density matrices (1 mg/ml), where average pore size is still smaller than the cell (2.3 μm) but fibers and protrusions are highly aligned, results in only a 50% reduction in cell invasion distance, indicating MMP independent motility and significant cell deformation occur. Yet, cells maintain a higher level of motility than expected based on previously reported steric limitations of pore size13. This could be due to differences in pore size measurement techniques or may indicate that fiber alignment influences cell deformability.
Overexpression of MT1-MMP in our study suggests that a threshold may exist for MMP utility, even in matrices of relatively small pore size. For cells embedded in low-density 1 mg/ml matrices with pores averaging 2.3 μm in diameter, cells move rapidly and invade further than in any other condition. In this case, overexpression of MT1-MMP did not result in any significant increase of invasive ability. Low ECM densities are known to reduce baseline MMP production while high ECM densities feedback to increase baseline MMP production38,39,40,41,42. This suggests that cells in low-density 1 mg/ml matrices could benefit from overexpression of MT1-MMP, but in fact do not. It is possible that overexpressed MT1-MMP in cells in 1 mg/ml remains inactive. Alternatively, because MMPs are most active in protrusions43,44, and protrusion number and spatial orientation about the cell body were modulated by matrix architecture, it is possible that aligned fibers optimize the efficient use of MMPs by spatially and temporally focusing the activity over longer time scales. Or aligned fibers may present cleavage sites in a distinct and more susceptible way. Future work will aim to further delineate these mechanisms. It is interesting to note that in our previous work, we discovered that matrix-embedded cells lacking certain focal adhesion proteins display variations in cell protrusion rate, orientation, cell speed, and invasion, which resemble some of the same motility outcomes that were achieved here by simple variations in 3D ECM microstructure. This highlights the fact that physical changes in the ECM can affect the outcomes of cell motility as drastically as genetic changes in cells, and demonstrates the importance of characterizing and further understanding the role of the ECM in modulating cell motility.
This study shows that the ECM microstructural parameter of fiber alignment reliably predicts multiple features of cancer cell motility in 3D matrices. Fiber alignment modulates cellular protrusion rate and orientation, which regulate cell motility. Fiber alignment varies significantly with collagen density and the extent of matrix crosslinking, highlighting the need for standardizing ECM microstructure characterization, which may help unify outcomes across 3D motility studies. The techniques and results presented here lend further insight into the role of the physical ECM and crosslinking enzymes in cancer cell motility and metastasis.45
Materials and Methods
Cell Culture
HT-1080 cells (ATCC, Manassas, VA) were cultured in Dulbecco's Modified Eagle's Medium (Mediatech Inc., Manassas, VA) supplemented with 10% (v/v) fetal bovine serum (Hyclone Standard, Logan, Utah) and 0.001% (v/v) Gentimicin (Sigma, St. Louis, MO). Cells were passaged every 2–3 days for a maximum of 20 passages. MDA-MB-231 cells (Physical Sciences Oncology Center, NIH) were cultured in Dulbecco's Modified Eagle's Medium (Mediatech Inc., Manassas, VA) supplemented with 10% (v/v) fetal bovine serum (Hyclone Standard, Logan, Utah) and 0.1% (v/v) penicillin/streptomycin (Sigma, St. Louis, MO). Cells were passaged every 2–3 days. All cells were maintained at 37 oC and 5% CO2 in a humidified environment during culture and imaging.
MT1-MMP overexpression
Full-length human MT1-MMP (hMT1-MMP) was obtained by overlapping PCR and subcloned into the EcoRI/AgeI sites of pFLRcmv-Ypet-puro vector46. The vector was sequence validated prior to lentivirus formation and transduction. Localization and fluorescence indicate expression. Only cells fluorescent with MT1-MMP-Ypet were tracked.
3D collagen I gelation, crosslinking, and drug inhibitors
Cell-impregnated 3D collagen matrices were prepared similarly to that described previously1,4,47. Briefly, 20,000 cells suspended in a 1:1 (v/v) solution of culture medium and 10x reconstitution buffer were gently mixed by pipetting with the appropriate amount of low concentration rat tail type I collagen in acetic acid (BD Biosciences) on ice to achieve a 500 μl solution with a final concentration of 1.0, 1.5, or 2.0 mg ml−1 collagen. High concentration rat-tail type I collagen in acetic acid (BD Biosciences) was used for 2.5, 4.0, and 6.0 mg ml−1 conditions. Then, NaOH (1N) was added in the amount of 3.6% (v/v) of the volume of collagen to initiate polymerization. The mixture was again gently but thoroughly pipetted on ice. At this point, 0.1 μg (1X, 1 μl) or 1 μg (10X, 10 μl) of purified recombinant human Transglutaminase II (TGII) in solution (R&D Systems, Minneapolis, MN) was mixed into the solution for those matrices that were to be additionally crosslinked. Initial medium and reconstitution buffer volumes were adjusted equally to account for the additional volume of TGII. The solutions were mixed again thoroughly on ice, with care taken to avoid introduction of bubbles, and subsequently placed in a standard glass bottom 24-well plate. Immediately, the well plate was placed in a 37 oC incubator for at least 30 min. Additional warm cell culture medium, 500 μl, was then added on top of the matrices and the plate put back into a CO2 incubator for full pH buffering. Reproducibility of gel structures relied on consistently using the same dish size (24-well glass bottom plates) plus accurate volumes (good pipetting technique) to ensure reproducible gel thickness and pH, and consistent timing (mixing all ingredients together on ice in less than 30 sec) plus temperature control (keeping all ingredients on ice throughout the process, mixing everything on ice, and then immediately transferring to a temperature and CO2 controlled incubator for at least 1 hr gelation time). Since each of the above-mentioned variables can influence gel structure and because time is limited during the gelation procedure, quantification of matrix structure after the full process, using our image analysis methodology presented herein, served as our quality control checkpoint. Statistical analysis of data produced from the image analysis of matrix structure and from cell motility analysis demonstrates the reproducibility of our procedure and the utility of our image analysis method.
For MMP inhibition experiments, Marimastat (AnaSpec Inc., Fremont, CA) or vehicle control DMSO (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was mixed into the medium before it was added on top of the cell impregnated matrix to achieve a final concentration of 10, 20, or 30 μM in the well. Since DMSO itself was found to dramatically effect cell motility at low concentrations (data not shown), we note that making the final amount of DMSO in our experiments optimally minimal required the purchase of dry Marimastat to make high concentration stock solutions. The final concentration of DMSO which did not impair motility, as demonstrated by the vehicle control data, was 0.1% (v/v) DMSO in culture media. This value was used to back-calculate the appropriate mixture of Marimastat to DMSO to achieve the desired stock concentration of inhibitor. Low cell density in the matrices helped to ensure that cells moved as singlets and that motility measurements were accurate.
Immunofluorescence microscopy and reflection confocal microscopy
To visualize collagen fibers within a 3D collagen gel, both immunofluorescence and reflection confocal images were collected using a Nikon A1 confocal microscope equipped with a 60x water-immersion objective (Nikon) and controlled by Nikon Elements imaging software (NIS-3.1). Collagen matrices imaged with immunofluorescence were first gently detached from the well plate walls, then incubated with primary and secondary antibodies in PBS with 1% BSA for 24 h (Abcam ab24133 anti-collagen I and AlexaFluor secondary). For reflection imaging, the microscope was configured to capture 488 nm light reflected during illumination with the 488 nm laser. Reflection and immunofluorescence images of detached gels were used to validate the fiber analysis algorithm, but only reflection images of attached gels were used for correlation plots.
Cell tracking and motility
Cells embedded in 3D collagen matrices, far from the walls and bottom of the 24-well plate, were imaged at 10x magnification every 2 min for 16.5 h similar to that which was previously described1,4. Briefly, cells which were actively moving as singlets over the course of the timelapse were tracked using Metamorph software with the Metavue Track Objects Application (Molecular Devices, Sunnyvale, CA) with x and y position coordinates measured for each 2 min time point, the collection of points comprising the cell's trajectory. Cell speed was calculated by averaging the distance per 2 min time interval that each cell traveled. Net invasion distance is a measure of the maximum displacement of the cell from its point of origin. This was found by calculating the displacement of each x, y tracking point over 16.5 h from the cell's original x, y location at 0 h, then finding the maximum of these displacements for each cell. For correlation plots, min-max normalization was used to re-scale the average of each data set between 0 and 1, avoiding units bias in cross-comparisons.
Cell protrusion analysis
Orientation of cellular protrusions and number of cellular protrusions were calculated as described in detail previously1,4. Briefly, the position of protrusions at least 5 μm in length from the cell periphery were tracked and measured with Metamorph software (Molecular Devices, Sunnyvale, CA) and tallied by hand from time-lapse microscopy images taken at 2 min intervals. The space around the cell, originating at the cell's centroid, was divided into eight equal radial partitions with the anterior axis aligned with the longest initial cellular protrusion and fixed in this position. The orientation of protrusions was calculated based on the number of protrusions that were extended into each of the eight partitions over 12 hours. The polarization index, α, of cellular protrusions was calculated by equation 1,
where C2 is the number of protrusions produced along the lateral axis of each cell over 12 hours, i.e. quadrants 3 & 7 in the orientation radial plot (refer to references1,4). C1 (quadrants 1 & 5) is the number of protrusions produced along the anteroposterior axis of the cell over 12 hours, with the anterior axis being set by the initial polarization of the cell at time zero. In effect, this parameter measures the re-orienting ability of the cellular protrusion away from their initial orientation over 12 hours. A value near 1 means the cell remains highly polarized along its initial polarization axis, whereas a value near zero indicates that the cell protrusions explored every angle equally. A One-Way Anova and Tukey post-test were used to calculate significance.
Image pre-processing for reflection confocal collagen image
The non-uniform background, IB(x,y), in the raw image,(IR(x,y)) (Supplementary Fig. 2A), caused by interference patterns of the reflected incident laser light, was estimated by interpolating the intensity distribution as a function of radial distance from the center (IB(r)) of the image. The relation between intensity and radial distance from center of image was established from angular-averaged intensity at different radical distances. Raw reflection images were then normalized, IN(x, y), using the relationship in equation 2 below (Supplementary Fig. 2B), where < > is denoted for the mean value.
Further, we enhanced the fibrous structure in reflection confocal image by applying a fiber enhancement filtering (FEF) algorithm. The procedure for FEF in brief is stated below. At first, a mask filter (MSKθ) with size of 5 pixels by 5 pixels is created with a line across the center line at orientation θ. A 2-D anisotropic Gaussian filter (GFθ) was obtained from the normalized product of the line mask and was the same size (5 by 5) of the Gaussian filter. A 2-D anisotropic average filter (AFθ, 5 by 5in size) was also obtained from the normalized product of the line mask. The raw image was convolved with these two filters respectively to access the filtered image FEFθ, which is the result of subtracting the average filter convoluted image from the Gaussian filter convoluted image. FEFθ at 15 different orientations distributed incrementally from 0 to 180 degree was used and the fiber enhanced filtering image (IFEF) is obtained from the maximum intensity projecting of FEFθ (Supplementary Fig. 2C). The binary of FEF image (IBW) is obtained by setting a suitable threshold value (th) to the fiber enhance filtering image (IFEF) to highlight the collagen fibers (Supplementary Fig. 2D). The standard deviation for Gaussian filter was tested and found to be optimized at a value of 0.7. All image processing was conducted in MATLAB. All images were taken at the same magnification and size (1024 × 1024 pixels, 60x magnification) and were analyzed at the same size.
Collagen pore size analysis
Relative differences in the mean pore size for the different concentration collagen matrices were estimated from the binarized reflection confocal images (refer to previous section for image pre-processing details, Supplementary Fig. 2) using a sequential morphological open operation based on techniques described by Gonzalez and Woods48 (Supplementary Fig. 3). First, the inverse or conjugate of the binary image was created, i.e. equation 3 below, to highlight the void regions between fibers (Supplementary Fig. 3A).
This conjugated image was then subjected to a sequential morphological opening operation of increasing width using a disk shape (Supplementary Fig. 3B–D). After morphological opening with a disk shape with radius of r, the remaining pore area, which is the summation of intensity in the image, I(r), represents the pore size with characteristic size larger than r (Supplementary Fig. 3E). Therefore, the areas in the image with pore size of radius equal to r are estimated by equation 4 below.
The frequency distribution at different pore sizes within an image was approximated by this distribution, equation 5 below.
For each collagen concentration, the mean of the images' pore size distributions were calculated and plotted (Fig. 2). Then these values were normalized within the set of varying collagen concentrations and plotted in correlation graphs (Figs 2 and 3).
Collagen fiber alignment analysis
After pre-processing of the x, y reflection confocal images to enhance fiber detail (see "Image Pre-Processing…" section of Methods and Supplementary Fig. 2), the alignment of the fibers, i.e. the anisotropy of the white space in the resulting binary image (e.g. Supplementary Fig. 2D), was estimated using a discrete Fourier Transform (FT) method similar to that described by Sander and Barocas49. Broadly, this method measures the anisotropy (alignment) across the image of reflected light intensity peaks (fibers) after filtering and binarizing. Supplementary Fig. 4 shows example computer generated images and experimental pre and post-processed reflection images as well as their resulting polar FT plots and alignment values. FT allows the description of a spatially complex digital image to be represented in terms of the frequency of its components. The two-dimensional FT (Supplementary Fig. 4B,E) of a digital image of size M × N (Supplementary Fig. 4A,D) was calculated by equation 6 below,
where x and y are the spatial coordinates of the digital image, u and v the frequency components along x and y in the Fourier domain, and . The magnitude of FFT (FT(u, v)) is calculated from equation 7,
where R(u, v) and I(u, v) are the real and imaginary parts of FT(u, v). Then random transformation was applied to calculate the integrated intensity (FI (θ)) over a line through the center at different orientations in the FT image. This Polar distribution of integrated FFT power was then transformed to Cartesian coordinates represented by equation 8 below (Supplementary Fig. 4C,F).
The two eigenvalue (λ1 and λ2, where λ1 > λ2) of the matrix [CxyTCxy] are used to calculate the alignment index, α, as α = 1 − λ2 /λ1. A value of α = 1 represents a completely aligned matrix of fibers, and α = 0 represents an isotropic matrix of fibers.
Determination of elastic modulus
A strain-controlled rheometer with steel cone-and-plate geometry (25 mm in diameter; RFS3; TA Instruments) was used to measure viscoelastic properties of the varying concentrations of collagen matrices. First, collagen matrices were gelled between the cone and plate for 30 min at a temperature of 37 oC in a humidified environment. A dynamic strain sweep was performed from 0.1% to 100% at a frequency of 1 rad/s to determine the region of linear elastic behavior, where the storage modulus G' is independent of strain, i.e. below the critical strain where the network structure is disrupted. Three replicate experiments were performed for each collagen concentration, with new gels being formed for each replicate. Based on these results, a strain in the linear elastic regime for each collagen gel concentration was chosen (0.3% for 1–2 mg/ml, 0.8% for 2.5–6 mg/ml). Then new gels were made and a frequency sweep was performed at said strain from 0.1–100 rad/s. This was again repeated for three separate gels for each collagen concentration. The storage modulus was found to be independent of frequency in the range of ~6–100 rad/s for all concentrations, and this is the G' that is reported.
The number of cells and biological repeats (n) for each experiment are indicated in the figure captions. Mean values ± s.e.m. and statistical analyses were calculated and plotted using Graphpad Prism (Graphpad Software, San Diego, CA). One-way analysis of variance and Tukey post-tests or pairwise two-tailed t-tests were conducted to determine significance, depending on the number of variables compared. Significance (p) was indicated within the figures using the following scale: *** for p < 0.001, ** for p < 0.01, and * for p < 0.05. Linear regressions were calculated and plotted using Graphpad Prism to visualize degree of correlation in correlation plots. Slope, square of the Pearson correlation coefficient (r2), and significance (p) of the regression's deviation from zero slope and are shown on the plots.
Additional Information
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