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Museums commonly deal with old things and creatures that have long shuffled off the mortal coil. You would not immediately associate them with a holiday like Easter which celebrates new life. However, among the collections at Buxton Museum, there are a few peculiar eggs; traditional symbols at this time of year. We thought we would share some of them with you while we are closed for renovation.
Eggs made from rock, minerals and gemstones were popular in the Victorian and Edwardian eras. We can only speculate why. With no internet, the people of these eras had to resort to talking to each other so perhaps such novelties inspired cheerful conversation. Indeed, Buxton Museum still sells quite a lot of colourful marble eggs in its gift shop. They look pretty and feel pleasingly tactile in the palm of your hand.
This egg has been crafted from the local rare mineral called Blue John, mined in small quantities in the Peak District village of Castleton. It dates from the early 20th century and is 13cm long. Buxton Museum has all sorts of intriguing objects made from Blue John, all displaying the same unusual purple-blue-yellow colour from which it gets its name; bleu-jaune, meaning blue-yellow in French.
This is a close-up of a petrified birds' nest. Objects can be turned into limestone by exposing them to mineral-rich water or "petrifying them". This specimen is from the collection of Randolph Douglas who once had his own museum in Castleton, the same village where Blue John can be found. He gathered fascinating items from around the world and exhibited them alongside keys, locks and miniature dioramas of his own ingenious creation. Douglas had a passion for escapology; the art of breaking free from death-defying traps, which ultimately hooked him up with famous magician and escapologist Harry Houdini.
When Buxton Museum and Art Gallery reopens on Tuesday 6 June, you will be able to see brand new displays featuring both Blue John and Randolph Douglas; admission free! We hope to see you.


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Luca di Tomme: A Sienese Fourteenth-Century Painter
Luca di Tomme: A Sienese Fourteenth-Century Painter
Sherwood A. Fehm, Jr., analyzes the paintings, provides ecclesiastical and communal documents pertaining to both the artist and his art, and presents a complete catalogue to re-create the life and to explicate the art of Luca di Tommè, the leading painter in Siena after the Black Plague of 1348.
In this first full-length study of Luca di Tommè, Fehm revives a neglected yet important artist who flourished during a period previously overshadowed in art history. He identifies Luca's achievements and relates them to the rich tradition of Sienese painting. Placing Luca in the fourteenth-century Italian tradition, Fehm briefly outlines the careers of the four great Sienese painters of the first half of the century: Duccio di Boninsegna, Simone Martini, and the brothers Ambrogio and Pietro Lorenzetti, the artists who most influenced Luca.
In addition to the detailed catalogue of Luca's works, which is divided into Accepted Works, Doubtful Attributions and Shopworks, and Lost Works, Fehm establishes for the first time an accurate chronology. The study is completed with photographs of all of Luca's paintings, many of them taken specifically for this book. For close investigation, specific details from a number of the more important works are illustrated.

subset: commoncrawl


text: Member E-mail Form Thank you for visiting the DentaQuest website! We are available to assist you with your Health First Colorado Dental Insurance. Do you have a question about what dental services are paid for (dental benefits)? Use this form to send DentaQuest your question. What is your relationship to member?: Member Parent Guardian Other How may we contact you? E-mail: Phone: ( ) - Second three digits Last four digits Best time to call: Select subject for your message: General Inquiry Benefits Question Appeal or Claim Question Changing Dentist Help Finding a Dentist Need New Member ID Card Please note: Email that you send to us on this website is NOT protected. There is a chance that the information you have given us here could be looked at or taken by someone else using the internet during the time it is traveling to us. Do not send personal information. You should send only your first name and telephone number and we will call you.


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The fallout from the Pedal for Scotland event continued at the latest Linlithgow and Linlithgow Bridge Community Council meeting.
Scotland's biggest charity cycle event was held on Sunday, September 10 and despite improvements to the access in and out of the town from the east end, some residents were still unable to get out of their homes because of road closures.
At the meeting one resident said: "There are quite a lot of elderly in the town. My aunt stays in Philip Avenue and people in the street couldn't get out at all."
He added: "She's locked up for the whole day and that is just unfair. You can't block a whole street off, she's 88 and relies on her car."
Ron Smith, from the community council, said: "I think the balance is shifted too much in favour of the event."
Councillor David Tait added: "No one is really against the event but no one is in favour of the full closure of the roads.
"All of these things were picked up in the local area committee meeting after last year's event and could've been avoided."
This year around 50 to 60 bikes suffered punctures after tacks were scattered on Station Road, Whitecross and Linlithgow.
A Cycling Scotland spokesperson said: "We're sorry for the disruption this event causes residents and businesses and are doing what a charitable event reasonably can do to mitigate it."


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Captain's log Stardate: 80025.9 (Sept 25, 2015)
A dinosaur playground? I stumbled upon it after seeing a friend of mine posted it in his Facebook. He posed before a 3-meter foot tall Tyrannosaurus Rex. I notice that there's also some dinosaur eggs next to the T-rex. Next to it was a smaller Tyrannosaurus, likely portrayed to be its child.
Being a dinosaur lover since young, I suddenly become tempted into it and asked my friend on its location. Turns out that its located at Toa Payoh.
So few days ago, me and a friend of mine when to check it out. After a few mins of searching, there it was!
Its like stepping into Jurassic Park! Well…..not entirely. Hahaha! There's the 3-meter foot tall T-rex, with the smaller one next to it. There's also the dinosaur eggs which lies next to the T-rex. There's also a green platform in the shape of a Stegosaurus. There's no swings, slides, see-saws or monkey bars in this playground, except for the dinosaurs themselves.
Such a unique playground. Daring kids might try to scale up the towering T-rex. Or even ride on the small one. Such imaginative. Such creativity that can be played at this playground. Especially when you are a fan of Jurassic Park, this playground can be of better use =D
Some people may find it weird to have a dinosaur theme playground been built in such a quiet neighbourhood. But I find it interesting though. Where can you find such playground like this elsewhere in Singapore?
Location of this dinosaur playground is in front of Block 27, Kim Keat Avenue (Toa Payoh).
Not far away from the dinosaur playground was another unique playground. But carries a piece of memory for those who are born in the 80s and 90s. They will lived to tell by its iconic design of animal theme shapes.
But one of them always seem to stands out among the rest. Quite a popular design for playgrounds and a popular place that kids can enjoy for hours! Yes, its the majestic Dragon playground.
I remember back then how I used to love running along its "spine" or body of the dragon and going in-between them. And sliding down the slide which was connected with it. Yes, the slides. Unlike now, most of the slides were built in swirl patterns, where back then the slides were just one straight slide to the ground.
Then, there's the simple swings and see-saws. And not to mention, little plastic rides in the shapes of seahorse and pony for children to sit on. And yes of course, all were built within the sandy ground. Nowadays you hardly seen such playground surrounded with sand. They were replayed by rubber mats, and some comes with an exercise corner. Nowadays most playground were built in a shape of houses and some in a form of a castle. All comes in standard plastic-made play-sets.
So on the same day itself, me and my friend pay this childhood place of ours a visit. A wave of our childhood memories hit us and soon we found ourselves playing on it. It feels good to being a kid again! =D
I came to know there are few other places that such playgrounds with dragon-based design still exist. And few of them had their sandy ground been replaced by rubber mats. Kids nowadays don't know how fun its like back then, playing around the dragon. I remember I even built a sandcastle by using the sandy ground. Its so much fun!
Well, some things will change. I wonder if the last of these unique playgrounds were to be demolished one day to make way for newer playgrounds one day. Its really an iconic design, and it would be very sad for those who treasure it dearly as part of their childhood memories when they are gone one day.
Time will tell. Just hope they still remains where they are. Still, I'm glad that I managed to jump back in this old school playground of yesteryear once again and revive my childhood playtime =)
Location for this Dragon playground is along Toa Payoh Lorong 6. Its surrounding were an open field. You can spotted it if you pass by the area. There are few others, where one of them is located at Ang Mo Kio.


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Radical Publishing's Earp: Saints For Sinners #1
EARP: SAINTS FOR SINNERS #1 (1 of 3)
Creators MATT CIRULNICK & DAVID MANPEARL
Writer M. ZACHARY SHERMAN & MATT CIRULNICK
Artist MACK CHATER, MARTIN MONTIEL and KYUSHIK SHIN
Colorist KYUSHIK SHIN
Layout Artist J.K. WOODWARD, ROB PEREIRA and JOE BENITEZ
Cover Artist ALEX MALEEV
Bi-Monthly Miniseries
Mature Readers
From Matt Cirulnick and David Manpearl comes a modern-day reimagining of a classic western hero.
In a world where the American economy has all but collapsed to the levels of the Great Depression, infamous bandits roam the country and the law is as corrupt as the criminals its sworn to stop. Yet one lawman remains a steadfast moral compass for the people: WYATT EARP. Earp has collared more most-wanted men than anyone in history – but after a violent assignment claims the life of his brother, Wyatt sets out to forge a simple life in the only boomtown left: Las Vegas. With gorgeous women and free-flowing money on endless tap, Sin City attracts more people than a modern gold rush. Though Earp no longer wears a U.S. Marshall's badge, his past is about to catch up to him. With nearly everything to lose, Earp will have to beat the odds stacked against him in order to bring old-fashioned justice to Sin City.


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The virtue of efficiency often goes unquestioned. Each of us has been given a calling. We are commanded to be diligent.
Sometimes though, efficiency can become too much of an ideal, at least for project-driven people like me.
I want the laundry folded and dinner made as fast as possible. I want to cross one more thing off my to-do list or shave a few minutes from my cleaning schedule.
Follow me over to Walking Redeemed to read the rest.
Linked up at Teach Me Tuesdays, Domestically Divine, Titus 2sdays, Encourage One Another, Homemaking Linkup, Women Living Well & Let's be Honest


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Best Ad Management Plug-in
After testing several ad management plug-ins, we came across this one and breathed a sigh of relief! None of the others provided the customization and tools we needed. We thought we were going to have to hire someone to code a plug-in from scratch.
There's a bit of a learning curve but once you've figured out how to use this, it's very easy and fast. We'll be upgrading to the pro version, this week.
The support is very good, too! We had a question about the plug-in and had an email answer within just a few hours. That's not typical when doing business between someone in the US and the Netherlands.
All-in-all, an outstanding piece of work. We highly recommend this plug-in and those who developed it.
- The topic 'Best Ad Management Plug-in' is closed to new replies.


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Fraser Valley Real Estate Board
15463 104 Avenue
Surrey, BC V3R 1N9
(604) 588-0325 (fax)
Monday to Friday: 8 am to 4:30 pm
Closed weekends and holidays.
Closed weekends and holidays.
The Fraser Valley Real Estate Board is an association of more than 3,400 real estate professionals who live and work in the Fraser Valley communities of Mission, Abbotsford, Langley, White Rock, North


text:
Open Monday to Friday 8:30 - 4:30
In Cobourg, we are all about shopping local. If you operate a business in Cobourg and don't already have a business profile, let us know and we'll provide you with details on how to create and manage your profile. This will allow you to perform updates at any time, add your logo and advertise your in-store promotions.
If You Have A Question Regarding the Business Directory
Contact the administrator at firstname.lastname@example.com or 905-372-5481 ext. 303.
Specializing in Leadership Entrepreneurial, Athletic, and Personal Coaching.


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Customs clearance
We utilise an Integrated Customs and Excise Support systems function through the network of logistics and managed by a crew of highly professional and trained personnel. This department effectively manages documentation, procedures and processes .The use of the EDI Gateway ensures reputable and efficient Services.We clear individual vehicles as well as containers. This way of shipping(via containers) is ideal for those purchasing four or more cars.
We have a specialized team that handle the import and export of both new and used vehicles. Our services are not limited to cars only, we clear heavy duty vehicles as well. This division offers a very efficient and personalized service to our many satisfied customers from all over Africa.
Our Services include:
- Processing of Customs Declaration "Bill of Entry"
- Payment of Port Charges
- Payment of Freight and Shipping Line Charges
- Handling a client's shipment from the time he makes his purchase until he receives delivery of his shipment.


text: Parenting is much more than controlling the behavior of your children. Parenting is actually about heart exposure and heart change. We know only Jesus Christ can create the kind of lasting change in the human heart that will transform behavior. Come and learn what it means to have the gospel shape what you say and do with the children who have been entrusted to your care.


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The value of $1
- Details
- Category: News
- Created: Thursday, 24 March 2011 05:36
- Last Updated: Thursday, 24 March 2011 05:36
- Written by Administrator
When service members purchase SGLI, in any amount, $1 of the premium ensures their eligibility for $100,000 in TSGLI coverage. This coverage applies to active duty members, reservists, National Guard members, funeral honors duty, and one-day muster duty.
Coverage became automatic Dec. 1, 2005, ensuring every member with SGLI also has TSGLI.
TSGLI payments are designed to help traumatically-injured service members and their families with financial burdens associated with recovering from a severe injury. Payments range from $25,000 to $100,000 based on the qualifying loss suffered.
For example, if a service member is hospitalized for 15 consecutive days after a traumatic injury, and the service member has basic SGLI coverage, compensation is $25,000. If four fingers on the same hand are lost due to a traumatic injury, the service member can receive $50,000. If sight in both eyes or hearing in both ears is lost due to a traumatic injury, compensation is $100,000.
It is important to note that this benefit is not the same as VA compensation for service connected disabilities. VA compensation for a traumatic injury doesn't automatically qualify the service member to receive TSGLI compensation.
The TSGLI benefit is also provided retroactively for members who incurred severe losses as a result of a traumatic injury from Oct. 7, 2001, to Nov. 30, 2005, if the loss was the direct result of injuries incurred in support of Operations Enduring Freedom or Iraqi Freedom.
There are certain injuries and conditions that are excluded from coverage. By law, TSGLI does not cover mental or physical illnesses or diseases, such as Post Traumatic Stress Disorder.
For more information or to determine eligibility, service members should contact their installation Casualty Assistance Representative or go to the VA website on TSGLI benefits, http://www.insurance.va.gov/sglisite/tsgli/tsgli.htm. Installation CARs can facilitate the claim submission process.


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Will Beatty said he doesn't keep track of individual game statistics, so he couldn't be sure if he'd ever before been part of an offensive line that gained a woeful 23 yards rushing on 19 carries as the Giants did Sunday in a 41-23 loss to the Broncos. But there are some numbers he is aware of.
"I know I've been a part of offensive lines that got running backs 1,000-plus yards [in a season], and I plan on doing that again,'' Beatty said. "With a slow start.''
The 23 rushing yards against the Broncos were the Giants' fewest since they had 6 yards at the Los Angeles Rams on Nov. 12, 1989, in a 31-10 loss.
According to Pro Football Focus, an analytical website, the Giants' running backs had 24 rushing yards after first contact with a defender. In other words, had they not broken tackles, they would have finished with minus-1 rushing yards for the game.
"I think we are close,'' Eli Manning said. "It's not like everybody is just messing up and we're getting out-physicaled. We are very close.''
The offensive lineman agreed.
"We have a fun uphill battle, but the season is far from being over and in no way are we saying we can't run the ball,'' Beatty said. "Yeah, we know we can make mistakes running the ball. So we get that out of the way and we move forward.''
Rolle call
Defensive captain Antrel Rolle stated the obvious when he noted that the Giants need to improve. But as always, he said it with more flair than most would.
"We need to understand that and we need to get extremely better,'' the safety said. "Not just a little bit better, but a whole lot better.''
Rolle also questioned the spirit of the team.
"I don't think we're playing with the excitement, the enthusiasm,'' he said. "What it looks like from the sideline, and outside looking in, it seems like sometimes we're in it, sometimes we're not.''
Giant steps
Tom Coughlin canceled his news conference Monday to tend to a family medical emergency. He was with the team throughout the day . . . The Panthers announced that safety Charles Godfrey is out for the season with a ruptured Achilles . . . Former Jets safety Kerry Rhodes reportedly will have a workout with the Giants on Tuesday.
We're revamping our Comments section. Learn more and share your input.


text: randomposting (randomposting) wrote, 2005-11-15 14:47:00 randomposting randomposting 2005-11-15 14:47:00 Previous Share Flag Next Mood: grossed out Music: Chatting. ... That's icky. There are recorded cases of babies that have been delivered through the rectum. In rare instances, blockage of the mother's vaginal orifice may force an unborn child into the rectal area and the baby must be expelled through the anus. A nineteenth-century British doctor named Payne cited the case of a thirty-three-year-old woman who gave birth in this manner. Dr. Payne, seeing that the child was located near the rectum, anesthetized the mother and delivered the child by forceps with little trouble. Subscribe Post a new comment Error We will log you in after post We will log you in after post We will log you in after post We will log you in after post We will log you in after post Anonymously switch LiveJournal Facebook Twitter OpenId Google MailRu VKontakte Anonymously default userpic When you submit the form an invisible reCAPTCHA check will be performed. You must follow the Privacy Policy and Google Terms of use. Post a new comment Preview comment Help 133 comments Post a new comment 133 comments


text:
Well, it's a little after 10:30pm. I'm where I usually am, on my bed with the dogs, watchin the recorded Waltons of the day. I'm still dealin with bronchitis, chest is feelin really tight, probably shoulda went in to doc today…will Monday if not significantly better I think.
I had a pretty okay day today. I forced my pain riddled body to get up and pull on some jeans and a tshirt, and my "Crazy Dog Lady and Proud of It!" Hoodie, and crawled into the van and drove up to our little lake cottage. It was a sunny day, but cold. The drive was nice, there are still beautifully colored leaves on some of the trees! It's only about a 40 minute drive, I was pretty sluggish on the way there. My mission was to get to the cottage and clean out the fridge and pantry for the season. I've been wanting to get that done since the end of August, but just havn't felt up to it.
So I got there and threw out anything bad, scrubbed out the fridge, then loaded the good stuff and the bad stuff (lettuce in a bag for a couple months = gross) into the van. By now my blood was kinda pumpin, so I thought I would hit the Dollar Store on my way outta town to get a few groceries, and I did…including milk and eggs! It saved me a trip to the "real" grocery store…alot smaller area to cover. I am sure I looked a fright considering I didn't comb my hair or put on makeup and was a bit dirty from the fridge…but…oh well!
Got my purchases loaded up, climbed in and started for home. The day was still sunny, the drive so pretty…alot of farmland, cows, horses…it made me sit there and thank God for all the beauty he created…I truly said thank you for each thing outloud as I drove!
Then I decided to put in my fave rocker chick (well maybe that's a tie with Joan Jett)…Pat Benetar! So for the rest of the drive it was me and Pat belting out her best work! Yes, it hurt my belly and lungs…but I held the wheel with one hand and my belly with the other and I sang my heart out! Of course "Hit Me.." got played more than once…cuz that's how I feel when I'm fightin…bring it! I've not been fightin much lately…been in the corner with the icky spit bowl droolin like a fool. I'm gonna put my mouth piece back in and go back in the ring again. I might get sidelined again here and there…of course I will…I have too many serious health conditions not to, but I will keep standin back up, I'm like that big blow up punchin bag clown we had as kids…lol
So, I enjoyed the day for the most part. I'm suffering a bit extra for it, but I'm okay. It was Veteran's Day and I also thanked God for all the Veteran's and prayed for their families, and thanked God mine was safe too.
Well, my chest is feelin pretty tight and icky, think I will break out the Vicks and take a bath in it. Hope you all enjoyed your personal 11-11-11!


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Ni no Kuni: Wrath of the White Witch – The Music of Joe Hisaishi
Namco Bandai has released the final behind-the-scenes look at the making of Ni no Kuni: Wrath of the White Witch. In this video, Joe Hisaishi, the composer and orchestral conductor that arranged all of the music, takes us through his involvement in the early stages of the project and the inspirations he drew from to develop the soundtrack. Ni no Kuni: Wrath of the White Witch will be available on January 22nd exclusively for PlayStation 3.


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Open Access This article is
- freely available
- re-usable
2012, 13(11), 14992-15011; https://doi.org/10.3390/ijms131114992
Betulin Complex in γ-Cyclodextrin Derivatives: Properties and Antineoplasic Activities in In Vitro and In Vivo Tumor Models
Faculty of Pharmacy, University of Medicine and Pharmacy, 2 Eftimie Murgu, Timisoara 300041, Romania
Organic Macromolecular Chemistry, Saarland University, Campus Saarbrücken, Saarbrücken D-66123, Germany
Institute of Pharmaceutical Technology, University of Szeged, 6 Eotvos ut., Szeged H-6720, Hungary
Faculty of Medicine, University of Medicine and Pharmacy, 2 Eftimie Murgu, Timisoara 300041, Romania
Author to whom correspondence should be addressed.
Received: 24 August 2012; in revised form: 23 October 2012 / Accepted: 24 October 2012 / Published: 15 November 2012
Given the present high incidence of melanoma and skin cancer, interest in potential drugs of plant origin has increased significantly. Pentacyclic lupane-type triterpenes are widely distributed in plants, offering numerous pharmacological benefits. Betulin is an important compound in the bark of Betula pendula Roth and has important therapeutic properties, including antitumor activities. Its biological effect is limited by its poor water solubility, which can be improved by cyclodextrin complexation. The best results have been obtained by using a novel cyclodextrin derivative, octakis-[6-deoxy-6-(2-sulfanyl ethanesulfonate)]-γ-CD. The complexes between betulin and the previously mentioned cyclodextrin were analyzed by scanning electron microscopy (SEM)and differential scanning calorimetry (DSC) and pharmacologically evaluated in vitro (MTT and immunocytochemistry tests) and in vivo in C57BL/6J mice. The solubility of betulin is improved by cyclodextrin complexation, which creates a stable complex that improves the in vitro and in vivo properties of the active compound.
Keywords:melanoma; betulin; cyclodextrin; MTT; immunocytochemistry; C57BL/6J mice; antitumor activity
1. Introduction
The interest in the development of drugs of plant origin to treat incurable diseases, such as cancer and AIDS, has increased in the last few years [1,2]. Due to the high incidence of melanoma and skin cancer, studies of new therapeutic agents are highly important.
Pentacyclic lupane-type triterpenes are widely distributed in various species of plants (i.e., Betula sp.), and the most active are betulin (Bet) and betulinic acid (BA). In addition to the use as folk remedies by Native Americans [2], BA has been found to act against HIV [3] and melanoma [4]. BA can be easily obtained via oxidation from another major component of birch bark, Bet [5]; both substances have been intensively studied, revealing a great number of pharmacological benefits.
Bet, lup-20(29)-ene-3β,28-diol, is an abundant compound that can be easily isolated from the bark of Betula pendula Roth species. Bet plays an important role in the semi-synthesis of BA and other active compounds [5]. In addition, Bet has long been investigated for its therapeutic properties, specifically its anti-inflammatory and antitumor properties [6]. First, studies showed limited or no effects on cancer cells [7]. However, recent studies have demonstrated that Bet has an important cytotoxic effect on colorectal, breast, prostate and lung tumor cells [8] by inducing apoptosis [9]; therefore, it cannot be regarded as an inert precursor but as a valuable anticancer agent [9].
An important issue that limits Bet's biological effects is poor water solubility [5]. A possible solution to this challenge is the use of hydrophilic carriers, such as cyclodextrins (CDs). CDs are oligosaccharides derived from starch, consisting of toroidal shapes and hydrophilic outer faces, that form inclusion complexes with a large number of hydrophobic active drugs, which are accommodated in the inner cavity of CDs. Recent studies [10] have shown that pentacyclic triterpenes form inclusion complexes with natural and semisynthetic CDs, and the best results are obtained by using γ-CD derivatives, such as hydroxypropyl-γ-CD. However, all of these complexes show very low stability constants (K = 47 M−1), which impedes the delivery of highly potent drugs to a particular destination in the body. One possibility to increase the binding ability of CDs is to extend the cavity by the attachment of hydrophobic substituents. Steffen et al.[11] synthesized a library of substituted per-6-thio-6-deoxy-CDs, which complex with even highly hydrophobic molecules, leading to highly water-soluble complexes.
The present work focuses on the preparation and characterization of inclusion complexes between Bet and a novel CD derivative, octakis-[6-deoxy-6-(2-sulfanyl ethanesulfonate)]-γ-CD (GCDG) [12], which has the best solubilizing properties for Bet [13]. The complexes were then tested both in vitro and in vivo using experimental animal models.
2. Results
Scanning electron microscopy (SEM) was used to assess the microscopic aspects of the drug, the complexing agent and complex formation (Figure 1a–c).
Although this method does not completely confirm complex formation, it helps to identify the existence of a single component in the preparation products. In the SEM photographs, the pure drug, Bet (Figure 1a), is characterized by the presence of crystalline particles of a regular size and parallelepipedic shape. The physical mixture of Bet and GCDG showed a slight crystalline structure of both the drug and CD (not shown). Drug crystals mixed with complexing agent crystals adhered to the surfaces of each other. In contrast, a drastic change in the morphology and crystalline nature was observed in the 1:1 kneaded product (Figure 1c), revealing an apparent interaction in the solid state. The SEM photographs of the inclusion complexes prepared by the kneading method showed a characteristic morphology as the small-sized particles tended to aggregate, indicating the existence of an amorphous product with the presence of a single component in the complex, thus suggesting maximum complexation.
The differential scanning calorimetry (DSC) profile is depicted in Figure 2. For GCGD, the graph does not show any peaks within the temperature range of the study; however, its melting point started at 300 °C. Bet exhibits a single sharp endothermic peak at 260 °C, as determined by sublimation of the substance. The Bet-GCDG complex is similar to the CD curve, and the endothermic peak of Bet disappeared completely, indicating formation of a molecular complex.
2.3. MTT in Vitro Analysis
The complex activity was more intense compared with Bet alone in B164A5 cells, which can be observed in Figure 3.
Compared to the control, Bet and its complex with GCDG had important effects. Cyclodextrin alone had a small effect.
Note that the consecrated solvent used for testing active substances in vitro on different cell lines, DMSO, which is known for its neutral effects in a concentration less than or equal to 0.1%, had a very small effect on cellular viability (91% ± 0.11% viable cells). The results of the tested compounds from the assay were as follows: GCDG −88% ± 0.31% viable cells, Bet −52% ± 0.09% viable cells and Bet:GCDG 1:1 complex −62% ± 0.25% viable cells. The results are expressed as the mean ± standard deviation. One-way ANOVA followed by Bonferroni's post-test was used to determine the significant differences between various experimental and control groups; *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively, compared with the control group.
2.4. Defining Mechanism-Based Toxicity
The results of the determination of the mechanism-based toxicity are presented in Table 1 and SI and Figures 4–8 and S1. The stock solution and dilutions were used as presented in the "Experimental section" and also applied in MTT tests (Bet concentration, 10 μM).
According to Figures 4 and 5, GCDG has almost no effect on the melanoma cell line B164A5, as the percentage of afflicted cells showed no significant difference compared with control cells.
The Bet:GCDG 1:1 complex-treated cells slightly increased in apoptosis, while the dead cells were almost double the control, revealing that complexation induced a decline of the initial Bet effect. Bet-treated cells have an increased percentage of affected cells, which were almost equally divided between apoptotic and dead cells.
Apoptotic cells (sub-G0 phase) were present in a larger proportion when the cells were treated with Bet alone or GCDG:Bet 1:1 complex.
The B164A5 melanoma cell line viability assay using Annexin V/PI showed different percentage values for dead and apoptotic cells in the viability assay using Annexin V/PI, with Bet inducing the highest amount of cellular death (Figure 6a–d).
Addition of Bet alone to the culture media at a ratio of 1:300 also induced a marked decrease in the total cell number. When complexed with GCDG, however, Bet had a very small effect on cellular viability, but a large proportion of the cells were only propidium iodide (PI) positive (Figure 6c), indicating that this complex destroyed cellular membranes and exposed the naked nucleus of the cells.
Histograms of cell cycle analysis for B164A5 cells treated with GCDG (a), GCDG:Bet 1:1 complex (b), and Bet alone (c) compared with the control (untreated cells), are depicted in Figure S1 (supporting information). Seventy-two h after cellular treatment, less cells were adherent to the culture flasks, but the regenerative potential was maintained.
2.5. In Vivo Analysis
The tumor dimensions were visibly reduced after treatment with the Bet complex (Figures 9–11). The tumors were treated intraperitoneally with vehicle or with the Bet-GCDG complex in solution, and the tumor volume and weight were measured (Figures 10 and 11). The Bet-GCDG complex reduced the tumor volume significantly after 14 days of treatment, and the mean tumor weight was also drastically diminished.
Analyzing the in vivo data of the tumor dimensions during the 16 days indicated that GCDG-complexed Bet significantly reduced tumor growth.
VEGF and S100 expression (Figure 12) assessed in the in vivo data also indicated the positive activity of Bet in vivo. As mentioned in the Experimental section, uncomplexed Bet was not used for treatment because Bet alone is not soluble in water, and the aim of our research was to improve this deficiency.
The cord-like distribution of tumor cells (Figure 12a) differed from the compact appearance of the treated cells (Figure 12d). Note the predominance of pigmented cells in the second sample (Figure 12d, inset) compared with the first one (Figure 12a, inset). Staining for S100 protein was positive for both groups, but a more intense and homogenous reaction was registered for the first group (Figure 12b) compared with a moderate expression of S100 in the first line-group (Figure 12e). Additionally, differences in VEGF expression between the two groups were observed. Constant and homogenous VEGF expression characterized the first group with a cytoplasmic, granular pattern that was observed in most of the tumor cells (Figure 12c), while a low and inconstant expression characterized the second one, with a cytoplasmic, granular pattern in most of the tumor cells (Figure 12f).
3. Discussion
Bet is the most important compound in birch bark, which has been used in folk medicine for treatment of skin diseases [15], such as actinic keratoses and pre-carcinoma status of skin [16,17]. Important derivatives with an increased antitumor activity have been produced from Bet [15], which affects colorectal carcinoma (DLD-1), breast carcinoma (MCF7), prostate cancer (PC-3) and lung cancer (A549) [8]. In the case of A549 cells, apoptosis was induced [9]. Other studies revealed that Bet possesses low activity in several cell lines: melanoma (Mel-2), epidermoid carcinoma, leukemia and neuroblastoma [15]. The present study analyzed Bet antitumor activity in murine B164A5 melanoma cells and showed its therapeutic relevance, indicating that Bet can be effective in melanoma cells depending on their origin. Therefore, because Bet increased bioavailability, it could be used as a therapeutic agent in skin pathology.
The water solubility of pentacyclic triterpenes, such as Bet or BA, is very poor (only 0.02 μg/mL for BA) so they must be administered in animal models either via intraperitoneal or subcutaneous methods such as dispersed systems like nanoemulsions [18]. Changes in their formulation that enhance water solubility can increase bioavailability. Complexation with cyclodextrins is a well-known procedure to achieve this goal. Increasing the water solubility of these cholesterol-like triterpenoids by addition of hydrosoluble sections, such as sugar moieties at the C3 and/or C28 position of the lupane framework or synthesis of saponosides, led to potent pharmacological activity [19]. Natural and semisynthetic CDs have been involved in triterpene complexation [20] with good solubility but rather small stability constant values of the complexes, in which the best results were obtained with γ-CD derivatives. Higher binding constants in the range of 105–107 M−1 are necessary to deliver highly potent drugs to a particular destination in the body without dispersing along the way. One possibility to increase the binding ability of CDs is to extend the cavity by substituting the primary hydroxyls by thioether groups [12], with the resulting CD derivatives being capable of complexing with even very highly hydrophobic molecules [11]. These complexes are characterized by very high binding constants (>106 M−1) and are potentially applicable for chemo- or photodynamic therapy in cancer [21]. Our previous studies [13] have shown a significant increase in water solubility of Bet and BA by inclusion complexation with octakis-[6-deoxy-6-(2-sulfanyl ethanesulfonate)]-γ-CD (GCDG), with the binding constants reaching the value of K = 6 × 107 M−1.
Previous studies [22] have demonstrated the solubilization of birch bark extracts using beta-cyclodextrin derivatives without obtaining a real inclusion complex, possibly because the triterpene molecule was too large to fit in the beta-cyclodextrin cavity. The external association between triterpenes and cyclodextrins increases water solubility for the active substances, but the best results are achieved by preparing real inclusion complexes. Therefore, our study works with a gamma-cyclodextrin derivative, which has an inner cavity that in our opinion allows the entrance of the triterpene molecule.
DSC analysis offers direct information about the thermal stability/phase transitions of CD complexes [23]. In combination with other methods, DSC analysis provides experimental evidence for the formation of CD-drug complexes and their stoichiometry in solution or solid state. DSC was used by Naidu et al.[24] to confirm the formation of meloxicam-CD binary systems. Similar conclusions were drawn by Mura et al.[25] in the study of CD-naproxen products. Liu et al.[26] used DSC to analyze CD complexes of an anticancer drug paclitaxel. In our previous studies, we also used DSC to characterize triterpene-CD complexes [27].
The endothermal peak of Bet indicates a sublimation process characteristic for the compound, previously confirmed by other thermal techniques [28]. The disappearance of the sublimation peak in the presence of the GCDG indicates that the active substance has been trapped in the cavity of the CD, and the sublimation properties are completely blocked in the studied temperature range. This behavior is direct evidence of the formation of a real inclusion complex.
Bet exhibits an important activity in B164A5 cells. Bet binds to the melanocortin receptors and promotes cell death in mouse melanoma cells [29]. Bet is still being investigated as an antitumor agent.
The B164A5/C57BL/6J mouse melanoma model has been used to evaluate chemotherapeutic agent activity because of the effects on the skin. B164A5 cells can be cultured in vitro and then transferred to the mouse body, specifically in C57BL/6J mice, which shows a high degree of acceptance. The inhibition of proliferation of B16 cells in the mouse, including tumor dimensions and histological evaluations, can be an indicator of treatment efficacy. Previous studies used B16 cells and C57BL/6J mice to test active compounds, such as dietary glycine [30]. A procedure was used in which the cells were first cultured and prepared in vitro in a proper medium, and then they were inoculated and transferred subcutaneously into the mouse [30]. The cell concentration is generally 105 or 106 mL−1, which leads to a model of tumor metastasis in a few weeks. A proper and reproducible model is helpful in testing pathological progression and the activity of chemotherapeutic agents. The aim of the present study was to obtain an experimental melanoma model and to evaluate the pathology after the application of a new formulation with Bet.
Figure 12 presents the progression of melanoma in vivo after the application of treatment (Bet and GCDG) at the end of experimental day 16. The HE images indicate a reduction in melanoma progression in the treated group. In the untreated group (group 1), two patterns of cutaneous melanoma were detected compared to the treated group (group 2) where there is only one pattern. In group 1, multiple necrosis, an oval tumor with a clear delineation and compact carcinoma beaches with vascular structures lacking endothelium and pigmented cells, was present. Group 2 was characterized by a reduced volume of cutaneous melanoma and no sign of affecting neighboring organs due to invasive cells. S100 and VEGF expression was more reduced in group 2 compared to group 1.
The caspase activity was upregulated neither by Bet nor by the complex suggesting a different mechanism of tumor cell death. Betulin derivative compounds upregulate caspases 8 and 9 in the HT-29 colon cancer cell line, which was not tested for Bet activity alone [31]. In addition, Bet-induced apoptosis does not involve the death receptor pathway but is dependent on mitochondria. Cytochrome c release and caspase activation occur independently of the Bcl-2 family proteins. [9]. Some previous studies [32,33] have shown that Bet apoptotic activity involves the sequential activation of caspase-9 and caspase-3/-7 and the cleavage of poly(ADP-ribose)polymerase (PARP). In another study [34], Bet pretreatment inhibited cadmium-induced apoptosis through an incomplete blockage incaspase-9 or -3-activation. Our study has clearly demonstrated that the Bet mechanism of action does not involve caspase-2 regulation.
C57BL/6J mice and others that have a common strain, B6D2F1, were used for a syngeneic tumor model with B16F10 cells [30,35]. The B16 melanoma model has been extensively used to analyze therapeutic agents for impact in this pathology [30].
Even though Bet is not as extensively used in melanoma treatment as BA [36], the present study claims its applicability as a therapeutic agent in this pathology if it is applied in a hydrophylic formulation. Application of the active compound at the beginning of the experiment for subcutaneous development of pathology determines an important reduction of pathological effects at the skin level. There are also some studies in which birch bark extract (with Bet as main component) was applied on mice with induced mutagenesis [37] or was analyzed pharmacokinetically [38]. Bet, like other pentacyclic triterpenes, is also important due to its lack of toxicity [38]. Because both the in vitro and in vivo activities of Bet are more effective as a GCDG complex, the mechanism of action likely occurs via direct action. Generally, small tumors (<2 mm diameter) are not dependent on angiogenesis. After experimental day 16, the tumors of the untreated group were larger than the ones of the other group. In addition, B16 tumor growth in vivo is dependent on vascularization. Tumor growth is slow and linear before vascularization but rapidly increases after blood vessel contribution occurs [30]. This tumor growth indicates a possible inhibition of neovascularization and the anti-angiogenic in vivo activity of Bet. Additionally, previous studies on embrionated eggs confirmed the anti-angiogenic character of Bet [39].
A possible mechanism of action of Bet may be its transformation into BA at the cellular level. This transformation may occur in the mitochondrial microenvironment, which contains several oxygen radicals that could induce Bet oxidation. Entrapment into cyclodextrins may influence this transformation. Future studies may quantify both plasmatic Bet and BA using electrospray liquid chromatography/mass spectrometry as previously applied for the BA alone [40]. VEGF and S100 expression were very intense for the untreated group compared with the treatment group, which supports the conclusion that Bet acts as an antitumor and anti-angiogenic agent as well as an antimelanoma agent. Tumor progression in terms of size and volume confirms the intervention of Bet on melanoma development. Proper formulations such as solutions are easily applied to the parenteral route. The preparation of solutions with an increased capacity to deliver an active agent in an emergency using fast routes, such as intraperitoneal methods, is an important goal of pharmaceutical technology. The high potency of Bet on the in vivo model illustrates its efficacy as a hydrosoluble agent in skin melanoma.
4. Experimental Section
Octakis-[6-deoxy-6-(2-sulfanyl ethanesulfonate)]-γ-CD (GCDG) was synthesized at the University of Saarland as previously published [11]. Betulin was purchased from Sigma Aldrich Ltd. (Taufkirchen, Germany, purity over 99%) and used as received. All other reagents were purchased and used as received.
4.1. Preparation of Complexes
Complexes were prepared using the kneading method: physical mixtures of Bet and GCDG were mixed with the same quantity of a solvent mixture consisting of ethanol and water (1:1). They were kneaded continuously until the bulk of the solvent mixture had evaporated; the mixture was then dried at room temperature (25 °C, normal atmospheric pressure) for 24 h, and then it was put in the oven at 105 °C for 7 h until constant weight was reached. The final product was pulverized and sieved through a 100-μm sieve. The binary products were prepared using a molar ratio of 1:1. The water solubility of the complex expressed as Bet concentration is 4.45 mM as previously determined whereas the cyclodextrin is highly soluble in water [13].
4.2. Scanning Electron Microscopy (SEM)
The crystal morphology was examined by scanning electron microscopy (Hitachi S4700, Hitachi Scientific Ltd., Japan). A sputter coating apparatus (Bio-Rad SC 502, VG Microtech, UK) was used to induce electric conductivity on the surface of the samples. The air pressure was 1.3–13.0 mPa. The samples were fixed onto a metallic stub with double-sided conductive tape (diameter 12 mm, Oxon, Oxford Instruments, UK). Images were taken in secondary electron image mode at 10 kV acceleration voltage.
4.3. Differential Scanning Calorimetry (DSC)
DSC was performed using a Mettler Toledo STAR Thermal Analysis System, DSC 821 (Switzerland). Argon was used as the carrier gas, the heating rate was 5 °C/min and the sample weight was 2–5 mg. Examinations were recorded from 25 °C up to 300 °C.
4.4. MTT in Vitro Analysis
The B164A5 cell line (ECACC; Sigma Aldrich origin Japan stored UK) was seeded onto a 96-well microplate and attached to the bottom of the well overnight. After 24 h, 200 μL of new medium containing Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL, Invitrogen, Carlsbad, CA, USA) and the tested substances were added and incubated for 72 h; the medium was supplemented with 10% fetal calf serum (FCS; PromoCell, Heidelberg, Germany) and 1% penicillin/streptomycin mixture (Pen/Strep, 10,000 IU/mL; PromoCell, Heidelberg, Germany). Melanoma cells were passaged at confluence after treatment with 5 mM EDTA; the living cells were then assayed by the addition of 20 μL of 5 mg/mL MTT solution. The intact mitochondrial reductase converted and precipitated MTT as blue crystals during a 4-h contact period. The medium was then removed, and the precipitated crystals were dissolved in 100 μL of dimethyl sulfoxide (DMSO; Sigma-Aldrich, Ayrshire, UK). Finally, the reduced MTT was spectrophotometrically analyzed at 545 nm using a microplate reader; wells with untreated cells were used as controls. All in vitro experiments were performed on two microplates with at least five parallel wells. DMSO was used to prepare stock solutions of the tested substances, and the highest DMSO concentration (0.1%) of the medium did not have any significant effect on cell proliferation. The dilution rate was 1:1000, and the concentration of stock solution was 10 mM.
4.5. Cell Culture Immunocytochemistry
Immunocytochemistry was performed for B164A5 cells (ECACC; Sigma Aldrich origin Japan stored UK) plated at a density of 10,000 cells/cm2 in 4-well glass chamber slides (Nalgene Nunc International, New York, NY, USA) and expanded for 24 h in culture medium. Bet, GCDG and Bet:GCDG 1:1 complexes were added to the culture media so that the dilution ratio was 1:300 and 1:1000, respectively. The control well contained untreated cells. Cells were maintained in culture for 72 h after addition of the substances and then prepared for immunocytochemical staining. After removing the culture medium, cells were washed and fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) and then analyzed for the expression of the proteins of interest, such as anti-h/m Caspase 2 (mCaspase 2 affinity purified rabbit IgG) (R & D Systems, Abingdon, UK). Untreated cells were also stained for the expression of cytokeratin 10 (mouse monoclonal IgG1, Santa Cruz Biotechnology, USA) and E-cadherin (mouse monoclonal IgG1, Santa Cruz Biotechnology). The staining protocol used the secondary biotinylated antibody binding and substrate addition (AEC) (Dako EnVision™ + System-HRP, Dako, CA, USA) following the manufacturer's protocol. After counterstaining with hematoxylin solution (Hematoxylin, Mayer's Lillie's Modification, Dako) for 30 s and washing with tap water, the slides were mounted in an aqueous mounting media (Crystal/Mount™, Biomeda, CA, USA). Microscopy analysis was performed on a Nikon Eclipse E800 microscope.
4.6. Annexin V/PI Assay
Annexin V-FITC (Miltenyi Biotec, Gladbach, Germany) was used in cell death flowcytometric studies (apoptosis) combined with propidium iodide staining solution (BD Biosciences, San Jose, CA, USA) following the manufacturer's protocol. Briefly, 106 cells were washed in 1 × Annexin V Binding Buffer (BD Pharmigen), centrifuged at 300 g for 10 min, resuspended in the same solution and incubated with 10 μL of Annexin V-FITC for 15 min in the dark. After washing the cells with 1 mL specific binding buffer and centrifugation, the cell pellet was resuspended in 500 μL binding buffer, and 1 μg/mL of PI solution was added immediately prior to analysis by flow cytometry.
4.7. Cell Cycle Test
B164A5 cells (ECACC; Sigma Aldrich origin Japan stored UK) including control, betulin, GCDG-complexed betulin and GCDG-treated were submitted to cell cycle analysis using the CycleTest™ Plus, DNA Reagent Kit (Becton-Dickinson, San Jose, CA, USA). This method involves dissolving cell membrane lipids, eliminating cellular cytoskeleton and nuclear proteins, digestion of cell RNA, and stabilization of nuclear chromatin followed by propidium iodide (PI) binding to the clean, isolated nuclei. All procedures were performed according to the manufacturer's protocols and included a first step of addition of 250 μL of Solution A (trypsin buffer), which was incubated for 10 min at room temperature. For the second step, 200 μL of Solution B (trypsin inhibitor and RNase buffer) was added and incubated for 10 min at room temperature. For the last step, 200 μL of cold (2–8 °C) Solution C (propidium iodide stain solution) was added to the same tube, gently mixed and incubated for 10 min in the dark in the refrigerator.
Data acquisition for the Annexin V/PI assay and cell cycle flow cytometric procedures was performed on a four-color capable FACSCalibur (Becton-Dickinson) flow cytometer, and the data were analyzed with the CellQuest Pro software (Becton-Dickinson).
4.8. Syngeneic Tumor Model
Ethics Statement
The experiment was analyzed and approved by the Bioethics Committee of the University of Medicine and Pharmacy "Victor Babes" Timisoara, Romania. The work protocol followed all of the NIAH-National Institute of Animal Health rules: animals were maintained during the experiment in standard conditions: 12 h light-dark cycle, food and water ad libitum, temperature 24 °C, and humidity above 55%. At the end of the experiment, animals were sacrificed by cervical dislocation.
Male C57BL/6J mice (eight weeks old) were purchased from Charles River (Sulzfeld, Germany). Five males were assigned to group 1 and were treated with GCDG and solvent (saline solution), and a second group of five males (group 2) was treated with the Bet-GCDG complex. B164A5 (ECACC and Sigma Aldrich, origin Japan stored UK) cells were grown as described in the MTT assay protocol. For the subcutaneous injection with B16 cells in mice at a concentration of 0.5 × 105 mL of cells without media, saline solution was used. After two days post-cell inoculation, the Bet-GCDG complex was administered intraperitoneally at a dose of 20 mg Bet per body weight daily for 14 days. After 16 days the mice were sacrificed and the pathological area was analyzed. The dose of Bet was determined based on a corresponding dose of BA, a related compound. Tumor size was measured every day using a caliper, and tumor volume was calculated as described by Eichenmuller et al.[14]. Body weights of the two groups of mice were recorded throughout the experiment, and all mice were monitored during the whole experiment period. Because Bet is insoluble in water, it could not be used alone for such applications.
4.9. Histology, S100 and VEGF Expression
The skin samples analyzed by the haematoxilin-eosin (HE) technique demonstrate the presence of conjunctive-subepithelial skin carcinoma with the capability of dermis and adipose tissue invasion in the cancer group. The healthy skin shows a normal aspect with no major changes. For the treatment group, the only detectable changes were a decrease in hair follicle number, altered morphology of the skin layers and an increase in dermal collagen fiber density. For the cancer group, a VEGF and S100 expression analysis was performed, which indicated the pronounced intensity of VEGF and S100 expression on the sub-epithelial area of the skin and the decrease in VEGF expression distant from the epithelium.
4.10. Statistical Analysis
All experiments were performed in triplicate. The results were expressed as the mean ± standard deviation, and the differences among the means were evaluated using the one-way ANOVA test followed by Bonferroni's post-test, with the level of statistical significance set at <0.05.
5. Conclusions
Bet is an active compound in murine melanoma. The solubility of Bet is improved by cyclodextrin complexation; this stable complex augments the in vitro and in vivo properties of the active compound. An increase in hydrosolubility demonstrates an important improvement for Bet antitumor activity. By complexation with suitable cyclodextrins, a parenteral formulation with a rapid effect can be prepared. The reduction in tumor size after Bet application was attributed to its anti-angiogenic effect.
Supplementary Informationijms-13-14992-s001.pdf
- Conflict of InterestThe authors declare no conflict of interest.
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Figure 1. Scanning electron microscopy (SEM) pictures of: (a) betulin (Bet), (b) octakis-[6-deoxy-6-(2-sulfanyl ethanesulfonate)]-γ-CD (GCDG) and (c) Bet:GCDG 1:1 complex.
Figure 3. MTT assay showing the effects of GCDG, GCDG:Bet 1:1 and Bet on the viability of the B164A5 melanoma cell line. Viability is expressed as percentage of viable cells compared to the control (considered as 100%). DMSO was used to prepare stock solutions of the tested substances, and the highest DMSO concentration (0.1%) of the medium did not have any significant effect on cell proliferation. The dilution rate was 1:1000, the concentration of the stock solutions were 10 mM (Bet, GCDG, Bet:GCDG 1:1), and the Bet final concentration in the medium was 10 μM.
Figure 4. Percentage of apoptotic cells as shown by Annexin V/PI staining. The results are expressed as the mean ± standard deviation. One-way ANOVA followed by Bonferroni's post-test was used to determine the significant difference between various experimental and control groups; *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively, compared with the control group.
Figure 5. Percentage of dead cells as shown by Annexin V/PI staining. The results are expressed as the mean ± standard deviation. One-way ANOVA followed by Bonferroni's post-test was used to determine the significant difference between various experimental and control groups; *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively, compared with the control group.
Figure 6. B164A5 melanoma cell line viability assay using Annexin V/PI. (a). Control; (b) GCDG; (c) Bet:GCDG 1:1 complex; (d) Bet.
Figure 7. Expression of immunocytochemical markers in the B164A5 melanoma cell line. Although this cell line is a mixture of spindle-shaped and epithelial cells, they lacked expression of caspase 2 (a), cytokeratin 10 (b) and E-cadherin (c). Magnification 400×, scale bars 50 μm. Bet concentration, 10 μM.
Figure 8. Caspase 2 expression in B16 4A5 cells treated with GCDG (a), GCDG:Bet 1:1 (b), and Bet alone (c). Bet concentration, 10 μM. Although the cellular number was markedly decreased when cells were treated with Bet, the caspase 2 pathway was not involved in this cellular death mechanism. Magnification 400×, scale bars 50 μm.
Figure 9. Macroscopic images of (a) untreated and (b) treated group of C57BL/6J mice at 16 days post-B164A5 tumor-cell inoculation. Note the reduction of the tumor in the treated group.
Figure 10. Tumor volume (mm3) for untreated or Bet:GCDG 1:1-treated mice, respectively. A paired Student's t-test was used to compare the differences between untreated and treated mice; *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively, compared with the control group. For the subcutaneous injection with B16 cells in mice at a concentration of 0.5 × 105 mL of cells without media, saline solution was used. At 2 days post-cell inoculation, the Bet-GCDG complex was administered intraperitoneally at a dose of 20 mg Bet per body weight daily for 14 days. After 16 days, the mice were sacrificed, and the pathological area was analyzed. Tumor size was measured every day using a caliper, and tumor volume was calculated as described by Eichenmuller et al.[14]. Body weights of the 2 groups of mice were recorded, and all mice were monitored during the whole experiment period.
Figure 11. Tumor weight (g) for untreated and Bet:GCDG 1:1-treated mice. A paired Student's t-test was used to compare the differences between untreated and treated mice; *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively, compared with the control group.
Figure 12. Morphology and immunohistochemistry of the studied melanoma samples. (a) HE of the untreatedgroup (scale bar 100 μm); (b) S100 for the untreated group (scale bar 50 μm); (c) VEGF for the untreated group (scale bar 50 μm); (d) HE of the treated group (scale bar 200 μm); (e) S100 for the treated group (scale bar 25 μm); (f) VEGF for the treated group (scale bar 50 μm).
Table 1. Evaluation of the variation between cell cycle phases between control cells and B164A5 melanoma cells treated with GCDG, Bet and their 1:1 complex.
|Sample (10 μM)
|Cell Cycle Phases
|sub-G0 (%)
|0.49 ± 0.02
|79.65 ± 3.99
|6.56 ± 0.16
|10.01 ± 0.26
|0.73 ± 0.04
|76.08 ± 2.21
|11.54 ± 0.26
|11.65 ± 0.18
|0.91 ± 0.04
|79.42 ± 3.82
|8.54 ± 0.18
|8.54 ± 0.16
|Bet:GCDG 1:1 complex
|2.27 ± 0.05
|82.76 ± 4.01
|4.81 ± 0.15
|8.41 ± 0.17
|1.21 ± 0.03
|67.83 ± 3.75
|15.09 ± 0.31
|10.39 ± 0.27
© 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).


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Canvas Uncorked with Ready, Paint, Fire
August 11, 2018 @ 6:30 pm - 9:30 pm$30 – $45
Ready, Paint, Fire hosts "Canvas Uncorked" in Warren Station at Keystone! Come and learn the basics of acrylic painting and enjoy a glass of wine in this upbeat and casual painting party. Not artistic? No worries. You don't have to be a "Van Gogh" to make a fabulous masterpiece for your wall. The instructors are an inspirational blast and will lead you step by step as you make a creation that is uniquely yours. And, there are plenty of cocktails to stir up your imagination. With a few guided strokes and some liquid encouragement, you will be amazed at the artwork that YOU made. One, complimentary house wine or beer, canvas, paint and supplies included in the entry. No experience necessary. Bottled wine and cocktail specials are also in the mix.


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Are You Working Your Abs the Right (and Tight) Way?
You got your ass up for the gym (or skipped happy hour for it), are dropping dough to be there, and killin' it in your fly performance gear. So why screw yourself (and your bod) when it comes to the actual workout? Our fitness expert Cara Weaver demonstrates the do and don'ts when it comes to some basic core postures so you don't sabotage your skinny goals. (All photos taken in the lovely Flybarre room at Flywheel Atlanta, where you'll find these moves and so many others to transform your body.)
Ever feel intimidated in a fitness class or at the gym? All of us have been there, but nothing screams "I don't know what the F I'm doing here!" more than a plank or pushup that's a hot mess. Boost your gym confidence (and get better results while avoiding injury) by practicing correct form for these standard moves you'll find in most classes and training sessions.
The good ol' plank is what I call the FLAT TUMMY MAKER! It works your transverse abdominals as well as your rectus (six-pack muscles), helping to stabilize your spine and create a strong core (SCORE!). There are two variations of it:
Do it right:
Get into a high pushup position with your hands in line with your shoulders. Keep a neutral head and spine; think about a long straight line from your head to your toes. Focus on the feeling of pulling the belly button into your spine to support your lower back.
Letting your back curve up or dropping your hips isn't strengthening your abs and is putting unnecessary pressure on your shoulders and/or back.
Do it right:
Start on your knees with forearms on the floor, elbows below your shoulders. Lift your knees off of the floor and straighten your legs. Follow the same rules above with a straight spine and pulling your belly button into it.
Do it wrong:
You put pressure on the shoulders by lifting your hips higher (first photo). Don't allow the low back to collapse from fatigue by relaxing the abdominals and sinking towards the floor (second photo).
This move, found in Pilates and barre classes, works your lower AND upper abs, back, and even thighs! However, do it incorrectly and your lower back will be screaming at you.
Do it right:
Pull the abs in and flatten your stomach to create a crease in your hips for the "V" to form. Keep your back straight, chin up, and chest proud. Balance on your tailbone and practice lifting your hands straight out in line with your knees without compromising your form, then further by straightening your legs (if you can).
Do it wrong:
You'll feel pain in your hip flexors and lower back if you round out your back and strain your neck forward to hold you up. And don't lift your legs until you're ready!
You know 'em. You love (to hate) 'em. Crunches/sit-ups are the easiest and most popular form of abdominal work focusing directly on burning out and sculpting those six-pack muscles.
Do it right:
Place hands behind your head for support. Keep elbows out wide and chin up (imagine a tennis ball under your chin). Lift your head, neck, and shoulders off the floor straight up. And SLOW DOWN; the slower and more controlled the better. If you're lift your legs to work the lower abs more, keep them at a 90 degree angle.
Do it wrong:
Make sure not to pull your elbows in front of your face with your chin to your chest (or pull your legs in). And don't move too fast, using momentum to get up; you could strain something, plus you won't be working your abs.
This handles your stubborn lower abs like a boss. It's a tough one, so doing it right is mandatory or you will just get frustrated…or hurt.
Do it right:
Start on your back with your legs together extended straight up in the air, making a 90 degree angle with your body. Place your hands on the floor, below your sacrum (the bone at the bottom of your spine) with your shoulders and arms pressed firmly into the floor and belly button drawn into the spine. Keep your tailbone planted into the floor and lower your legs to 45 degrees. Lift your head and hold.
Do it wrong:
Don't arch your back just to hold your legs closer to the floor! You will no longer be working abdominal muscles. Instead, lift your legs a little higher, bend your knees, or put your hands closer to/under your butt for more support.
Do these moves right and you'll be getting tight! You'll feel your tummy muscles strengthening, instead of your back and shoulders aching and be able to increase reps/length of hold as time goes on.
It's a
sure core thing.
Want to get to know Cara? Check out her website to "make healthy happen" or take one of her Flywheel or Flybarre classes.
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As a young writer who frequently has no idea what she's doing, I found this both comforting and challenging. On one hand, I'm glad to know I'm not alone. On the other, I'm a little mad that apparently I won't outgrow or out-succeed my insecurities. Apparently, I have to make nice with them. Tame them with light and love, rather than shoving them into the closet.
So today, I'm going to share my biggest insecurity. The one that Satan most often uses against me, the one that most often keeps me from stepping into God's full plan for me.
My biggest insecurity is that I'm too intense, and I ask too much of people.
Truth is, I have no idea how he does. I'm demanding. I'm obsessive. I care about everything. A lot. Some nights we stay up long past a reasonable teacher's bedtime because I need to vent about feminism or theology or someone's emotional baggage. You know, all those good 1 am topics.
And, as my college roommates (and suitemates and classmates) can attest, I absolutely cannot stay out of people's business. If someone within a one mile radius of me has emotional baggage, I will find it, poke at it until I see tears, and then attempt to counsel that person to freedom. Worst of all, I will enjoy it immensely. I once helped with an inner healing ministry, and we did these day-long intensive events. People would be on the floor, bawling their eyes out, and I could barely contain my excitement. During one event, a woman came up to me and asked me how, with so much pain around me, could I possibly be smiling. I just shrugged and told her I liked the freedom that comes after the pain. But I walked away feeling like a freak.
See, it's all well and good to care about people, but there's a point where it gets messy. And I run about five miles past that point. I insist that people become their best selves, which is great in theory, but really really uncomfortable in real life. I've ruined more than one friendship over it.
When Satan whispers these half-truths in my ear, it completely shuts me down. I back away from helping people. I people-please when I should be challenging. I keep silent about issues that matter, afraid of being labeled a rebel-rouser or a Jezebel. I hide in my closet of shame.
The only way I've found to combat this is to bring these lies into the light. I confess the ways I've let them derail me, and ask God and others to forgive my lack of action. Confession isn't really in vogue in the church anymore, but I always find it freeing. When the words roll off my tongue (or today, my keyboard), I feel Jesus turning everything upside down again, making darkness into light, weakness into strength. When I'm honest with my community, it builds unity. We get to say, "Me too," or pray for each other or just hug it out.
It takes some guts and faith, but I think we can all find a safe place to confess our insecurities. For some, that's in our church or small group. For others, it's just with Jesus. For some of you, it could be right here in the comment section of this blog. But wherever and however we do it, we can always find ample light and grace in our savior Jesus, who bore all sin and sickness (including those flaws we're ashamed of and all the ways we don't measure up) to the cross, defeating them and rising up in us a new person—fully free and fully enough.
May you find that identity, and may you boldly pursue the life God has called you to live. In the light, away from shame, and buoyed up by a community of grace.
With much love (and hopefully not too much pushing),


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Peak Performance Training for an Alternative Health Care Provider
Let's face it, the current business world isn't for wimps. There's more competition, more changes, and more that needs to be done than ever before. For the alternative health care provider, the challenges increase even more with misunderstandings about who you are, what you offer, and the value you bring to your potential patients. With reality being what it is, if you want your practice to succeed it becomes critical for you to be "hardy" enough to withstand the storms modern business brings.
What is hardiness? My definition is that it's the ability to roll with whatever life has thrown at you and to keep moving forward. Recently I read the book, Unbroken: A World War II Story of Survival, Resilience, and Redemption, by Laura Hillenbrand. This book is a biography of Louis Zamperini, who was an Olympian when WWII broke out, heard the call of his country and joined the war effort. While on a rescue mission, his plane crashed and he set the world record for surviving on the ocean. Once out of the ocean, he was captured by the Japanese and survived the brutal treatment in their prison camps.
The story tells an intense tale of what hardiness looks like. I couldn't help being in complete awe of the resilience of this man who stood in the face of horrendous obstacles and found ways to not only go on, but also, after surviving, to forgive and make the world better because of his message about being hardy.
Comparing the hardiness of a true Olympian and war hero against the hardships of the current business environment makes the challenges of alternative health seem like there's nothing to complain about. But, since in reality that's not fair, let's see what of Louis' heroics could be applied to your practice.
1) Face current challenging situations with patience. If Louis could endure surviving on a lifeboat with two other men for 47 days and not complain, we can endure the challenges of technology and the constant change it throws into our marketing model.
2) Look for ways to always maintain respect in business. The captive American soldiers maintained their sanity through subtle resistance to the Japanese and their efforts to demoralize. Since most of us aren't in a dire situation, look for ways to behave that you'll never regret and that help you to always maintain your respect.
3) Find a way to let go of past hurts and move forward to a clean slate. If Louis, by the grace of God, could find a way to forgive his captors, who beat him day after day for years, and find a way to not let those experiences haunt him—then alternative doctors can find a way past government politics and employee problems and let go of business hurts.
Can you image what a hardy business owner could accomplish if he or she approached their practice using the Louis style—implacable patience with the challenges, maintaining respect for self at all times, and not letting the past define their present business efforts?


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Dot Dot Boo Black Dotty Cat Yardage by Me & My Sister Designs for Moda Fabrics
$ 3.25 $ 4.75
Black Dotty Cat yardage from the Dot Dot Boo collection by Me & My Sister Designs for Moda Fabrics
Yardage is offered in 1/2 yard cuts. More than one half yard will be cut as one piece.
A quantity of 1 refers to a 1/2 yard cut.
A quantity of 2 refers to a one-yard cut.
Collection: Dot Dot Boo
Designer: Me & My Sister Designs
Manufacturer: Moda Fabrics
Piece Count: 1/2 Yard continuous cuts
Material: Cotton


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Product Description
MKS Sylvan Track Pedal (black). MKS Sylvan Track racing pedal is a an affordable and reliable pedal to race the Track with. It is ready to fit toe clip and straps to and is a traditional ball and cone style pedal. This is once again a high quality Japanese pedal from the home Keirin Track racing.
Additional Information
Additional Information


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KPMG UK appoints Pat Race as a partner in its pensions investment advisory team.
KPMG UK has appointed Pat Race as a partner in its pensions investment advisory team. Pat joins KPMG from Mercer where as senior partner he fulfilled many roles including leading the firm's investment business.
Pat will join KPMG this month and will be based in London. He will work with Nick Evans' team on all aspects of pension investments, from strategy to fund manager appointments.
Nick Evans, Partner, KPMG investment advisory and trustee services, says "This new role demonstrates the growing strength of KPMG's pensions practice, as an increasing number of trustees and businesses seek our support to navigate difficult markets, continued low interest rates and mounting regulatory challenges. I am immensely proud that someone of Pat's standing in the pensions arena shares our enthusiasm and ethos and is eager to join our team."
Pat Race adds: "KPMG's team has been doing some really innovative work to help trustees and scheme managers ride out volatility, de-risk and better assess scheme liabilities. I look forward to working with the firm's clients to help them tackle these issues and deliver the best results for members in what is a challenging time for the pensions industry."
Notes to editor:
For further information please contact:
Angela Pink, KPMG Communications Executive
E: email@example.com
KPMG Press Office
About KPMG
KPMG LLP, a UK limited liability partnership, operates from 22 offices across the UK with approximately 13,500 partners and staff. The UK firm recorded a revenue of £2.07 billion in the year ended 30 September 2016. KPMG is a global network of professional firms providing Audit, Tax, and Advisory services. It operates in 155 countries and has 189,000 professionals working in member firms around the world. The independent member firms of the KPMG network are affiliated with KPMG International Cooperative ("KPMG International"), a Swiss entity. Each KPMG firm is a legally distinct and separate entity and describes itself as such.
Request for proposal


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Cithaeron or Kithairon (Κιθαιρών, -ῶνος) is a mountain and mountain range about ten miles (sixteen kilometres) long, in central Greece. The range is the physical boundary between Boeotia in the north and Attica in the south. It is mainly composed of limestone and rises to 1,409 metres (4,623 ft). The north-east side of the range is formed by the mountain Pastra.
|Highest point
|1,409 m (4,623 ft)
|38°11′03″N 23°14′57″E / 38.18417°N 23.24917°ECoordinates: 38°11′03″N 23°14′57″E / 38.18417°N 23.24917°E
|Attica, Greece
Boeotia, Greece
The range was the scene of many events in Greek mythology and was especially sacred to Dionysus.[1] In Euripides' Bacchae, Dionysus carries out his dances and rites with his bacchants, his priestesses, on Cithaeron.[2] Oedipus was exposed on the mountain, while Actaeon and Pentheus were both dismembered on its slopes. It was also the place where Heracles hunted and killed the Lion of Cithaeron.
In historic times, the mountain acted as a backdrop to the Battle of Plataea of 479 BC[1] and was the scene of much skirmishing before the battle itself. In later times fortifications were built both at Plataea and Erythrai as the mountain formed the disputed natural border between Athens and Thebes.
The people of Plataea also personified the mountain as their primal king: "But the Plataeans know of no king except Asopus and Cithaeron before him, holding that the latter gave his name to the mountain, the former to the river". (Pausanias 9.1.2)
- ^ a b Gardner, Ernest Arthur (1911). Encyclopædia Britannica. 6 (11th ed.). Cambridge University Press. p. 395. . In Chisholm, Hugh (ed.).
- ^ Euripides, Bacchae, 62-63: 'For my part I will go to the glens of Cithaeron, where the bacchants are, and take part with them in their dances.'
External linksEdit
|This article relating to Greek mythology is a stub. You can help Wikipedia by expanding it.


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How to Build Loyalty through Predictive Analytics and Personalization
Q. What are some of the challenges today's brands face when trying to build loyalty in the age of the empowered consumer?
Empowered consumers have access to an incredible amount of information right at their fingertips, can easily compare products and pricing across brands, and switch on demand without consequence. Faced with an abundance of choice, they often rely on emotions to reduce cognitive complexity and guide their purchase decisions. They also increasingly exercise their control when making decisions based on whether the brand's values aligns with their own personal belief systems. Consumers are savvy, can see through many of the tactics marketers have long relied on to build retention and have heightened expectations for how brands communicate with and serve them. This makes it extremely challenging for brands that focus on offering the right product or service, or relying on undifferentiated offers and programs to build loyalty with their customers.
Q. Why is it important for brands to build emotional loyalty in addition to behavioral loyalty?
Forrester's research shows that emotional connections are key to establishing meaningful connections with customers. In fact, emotion is one of the strongest unique drivers of loyalty metrics like retention, enrichment, and advocacy. And it permeates every stage of the customer life cycle – from how customers feel when they are discovering and researching a product to how they perceive their relationship with a brand. Understanding emotional loyalty means understanding the context and drivers that surround a customers' loyal behavior. Brands that strive to build both emotional and behavioral loyalty are more likely to end up with a loyal base that chooses to stick with brand rather than just feeling "stuck" with the brand.
Q. Why is data collected about loyalty program customers a "super sample" for marketers?
Loyalty programs provide consumers with an explicit give-and-get value proposition: in exchange for allowing a brand to access and track their transactions, behaviors, and preferences, they'll receive material incentives in the form of discounts, personalized communications, and other recognition. Forrester's data reveals that half of consumers join loyalty programs of brands they are already loyal to, and, customers enrolled in a loyalty program are more engaged than non-members. They are more likely to perceive a higher quality experience, consider a brand for future purchase, spend more with the brand, and recommend a brand's products and services to family and friends. In effect, loyalty program data offers brands with valuable insight into the behavior, journeys, and perceptions of their most engaged customers so that they can win in all kinds of customer moments.
Q. What do marketers need to consider when building loyalty programs that drive active participation?
Contrary to what many brands might think, building an effective loyalty programs starts with an understanding of the customer, not the material value proposition. First, marketers need to build a deep understanding of customers, their journeys, and their expectations. In short, they must take an outside-in approach to program strategy: rather than thinking about what they want a program to do for customers, marketers must think about what customers want from a program. Second, marketers must consider the objectives of the business including incremental sales and retention but also objectives like customer engagement, increasing customer referral, or data collection. Third, marketers must select program benefits that balance consumer expectations and their business objectives. The most successful loyalty programs include benefits that incent behavior through tangible rewards, deepen relationships with engagement and experience-based benefits, and show appreciation for the choice consumers make to continue their relationship with the brand.
Keep in mind that establishing a compelling value proposition is only the first step to program success. Marketers can't forget to actually market the program and its benefits to consumers! On average, consumers belong to nine loyalty programs so the competition for consumer mindshare is steep. Whether they're launching a new program or ensuring that an existing program maintains healthy enrollment and participation levels, marketers must create a plan — across touch points —to educate consumers about the program and its value both at enrollment and as the consumer participates over time.
Q. What are the challenges marketers face in terms of applying data insights from loyalty, and what are your recommendations for facing them?
Loyalty marketers collect a lot of data from loyalty programs — spanning campaign response data, behavior data, transactional data, and preference data — but struggle to use that data to improve the relevance of offers, communications, and other interactions. In fact, their top challenges revolve around personalization, managing data from multiple sources, and making sense of customer interactions across all touchpoints. Essentially, bridging the gap between data and insight they can use to impact future campaigns and strategy is a major challenge that continues to stymie marketers. To overcome these challenges, marketers we surveyed already note that they plan to increase investments in customer analytics capabilities. This is a good first step, but not the only option. Marketers that work with a third-party to manage and execute their loyalty strategies should push their loyalty provider to provide specific insights focused on "what they should do next" to improve a program's ability to deliver relevant offers and communications. Internally, marketers should consider implementing or refining governance processes that connect insight to action. This means establishing a cross-functional group or iterative process spanning marketing, loyalty, and existing insights teams to identify the types of insight needed, opportunities to apply those insights, and distribution models for those insights. For instance, marketers should share loyal customer interaction data with other teams so they can use it as an input to personalized customer experiences, product enhancements, or web design.
Emily Collins, Senior Analyst at Forrester recently hosted webinar with Emily Rudin, Chief Customer Officer at CrowdTwist, on the topic, "How to Build Loyalty through Predictive Analytics and Personalization."
Watch the playback here.


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Customer Service can make or break a small, online business. This provides an opportunity to use and improve strategies and to deepen the knowledgeIt is best manner a business analyst can develop their behavior expertise and private qualities The performance of most analysts improves extra time as their experience grows, however this may be heightened and accelerated if our if the organization operates a proper coaching or mentoring program.
Because the term business" may be interchanged with day-to-day operations in addition to the general formation of a company, the term is often used to point transactions relating to an underlying product or service. SFIAplus allows organizations to classify and benchmark their IT expertise and to coach and develop their groups to satisfy the defined ability requirementsAs a business analyst, this provides a foundation so that you can gauge where you're in opposition to the abilities and corresponding level of competence defined within the framework.
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Jika Anda memiliki nomor telepon bisnis dan pribadi yang berbeda, Anda dapat memiliki kedua WhatsApp Business dan WhatsApp Messenger terpasang pada telepon yang sama, dan mendaftarkan dengan nomor yang berbeda. Prospects who want to run Microsoft 365 Business in their organization must be running Windows 7, eight.1, or 10 Professional on their current desktops and don't depend on a domain controller for his or her group.
In addition, a business that needs to boost cash on a inventory market or to be owned by a variety of people will typically be required to adopt a specific legal type to take action. To use for PayPal Working Capital, your business should have a PayPal business or premier account for no less than 90 days and course of between $15,000 (or for premier accounts $20,000) and $20 million inside those 90 days or within any time period less than or equal to 12 months.


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Monroe College-New Rochelle 434 Main St New Rochelle, NY 10801 Phone: (914) 632-5400
General Information About Monroe College-New Rochelle
Monroe College provides professional, career oriented higher education to students from diverse backgrounds. We proudly offer access and opportunity to motivated students who desire to enrich their lives in a personalized and supportive environment. We provide caring and effective teaching and sustain faculty who are passionate, knowledgeable, and dedicated to student success. We build on these strengths to prepare graduates for successful careers.
Institutional statistical data was gathered from the U.S. Department of Education - Institute of Education Sciences, National Center for Education Statistics, 2009-2010.
Institution Type
- 4-year, Private for-profit
Special Learning Opportunities
- Distance learning opportunities
- Study abroad
- Weekend/evening college
Student Services
- General Education Courses
- Career Services available upon completion
Credit Accepted
- Dual credit
- Credit for life experiences
- Advanced placement (AP) credits
- The acceptance of credits for work experience or military experience are subject to review by the school and cannot be guaranteed.
Religious Affiliation
- Not applicable
Federal Aid
- Eligible students may receive Pell Grants and other federal aid (e.g. Direct Loans).
Degrees/Certificates offered
- 1-2 years certificate
- Associate's degree
- Bachelor's degree
- Master's degree
Campus Setting
- Suburb: Large
Disability Services Student
- 3% or Less
Admission Total Applicants 1,308 Percent Admitted 59% Men Admitted 58% Women Admitted 60% Full Time Retention Rate 71%
Student Demographics Total Enrolled 2,621 Native American 2.0 Asian 188.0 African American 1,545.0 Hispanic 474.0 Enrolled FullTime Undergraduates 1,776 Enrolled FullTime Graduates 133