text:
※ This item is only Ear ring. Impossible Kai clip-on earwax ceremony. ※ post thickness 0.8mm (rough first base) petition attention one down. size: length about 4.5 cm material: natural zircon, pink tourmaline, hematite parts: All 14k gold filled A color that makes you feel like autumn with a bubble wrap and fruit, In natural brown of champagne brown Pink tourmaline's color point is the point Warm color earrings. Also put texture metallic hematite I made it sharp. The shape is simple marquis hook and it is easy to use and comfortable to wear. From everyday use to going out A feminine item with a simple line! * Natural zircon is a rare natural stone that is classified as a mineral, unlike cubic zirconia (CZ), which is a synthetic stone. Zirconia with a very high refractive index next to diamond is a great brilliance. It is also recommended for gifts and gifts at 14kgf all! Wireline is strong and stylish We use square wire that we have. (The cross section has a square shape) Most of our earrings are about 0.7mm thick It will be a piercing post. This item is slightly thicker at 0.8 mm, so if the piercing hole is not stable, Please note that the condition of the hall is not good. <Size> Approximately 4.5 cm in total length <Material used> Natural zircon AAA (not for synthetic stone cubic zirconia CZ) Pink Tourmaline AAA Hematite AAA <Use parts> All 14k gold filled ※ We deliver nano jewelry coating so that long-term better condition can be maintained. ※ There may be a small scratch because you are using a tool. Please acknowledge. -About shipping- Because I live in Vietnam, I will ship from Vietnam by international airmail. (2 weeks from date of shipment) Please read before purchasing ・ AN (Japanese) handcrafts one by one. -Please acknowledge that there are some differences between the photo and the real item for handmade products. ・ Color may look slightly different depending on the terminal you are viewing. ・ Because it is delicate, please be careful about handling. ・ Natural stone may show tints of color, inclusions and cracks, but please understand it as the personality of natural stone and its attraction except for obvious defects. <How to care> After use, wipe gently with a soft cloth that does not have an abrasive. After that, it is recommended to store it in a bag with a zip. Produced by Japanese / Made in vietnam
- 付款方式
- Alipay 支付宝, 添加信用卡快速付款, 输入信用卡卡号付款, PayPal, LINE Pay
- 退款换货须知
- 点我了解设计馆的退款换货须知
- 问题回报
- 我要检举此商品


text:
Join Our Mailing List
7-Eleven Making A Splash With USDA-certified Organic, Cold-Pressed Juices (2018)
The National Association of Convenience Stores predicted that healthier options would be one of the industry's biggest trends for 2018 and 7-11 wasted no time not just proving them right, but aiming to blow away the competition. America's largest convenience store chain has announced their new private label "7-Select GO!Smart" beverage which is not only organic, but each of the four varieties also promises to contain certified fair trade, Non-GMO Project verified, 100-percent fruit and vegetable juice, not from concentrate, with no additives and no added sugar.
They will offer all this in 14-ounce bottles for a $2.99 retail price. Flavors included at this time are:
- Clean & Green (made with kale, cucumber, apple, spinach, mint, celery, lime, and parsley)
- Tropical Glow (made with pineapple, orange, banana, apple, mango, passionfruit, and coconut water)
- Berry & Bright (made with tart cherry, carrot, blackberry, pomegranate, bilberry, cranberry, and acai)
- Restoration Red (tomato, tart cherry, beet, strawberry, apple, and lime

subset: commoncrawl


text:
Final draft of India's data privacy law may be submitted today
According to three members of the committee, a "middle ground" has been found on most of the contentious issues and the final draft of the much-awaited report is ready to be submitted.
25% ATMs of public sector banks may be vulnerable to fraud
ATMs run by PSBs may be vulnerable to frauds as 74% of cash dispensers are running on outdated software.
Parliamentary panel debates on summoning FB brass
Dr Gulshan Rai, the head of country's cyber security, a senior officer from the Intelligence Bureau among others were also present to give govt's view point.
Parliamentary panel pushes for data protection legislation
The Parliamentary Standing Committee on Finance in its draft report has urged the government to bring data protection legislation before Parliament at the earliest.
1000 emails in three hours against Aadhaar linking from new petition website
Number of emails sent crossed the 1,000 mark in little over three hours of the website going live, said those who started it. At least 780 of these were sent to MPs.
Rise of digital transactions a blip: Rajan
Rise of digital transactions during the demonetization exercise was a blip on the radar, said former RBI governor Raghuram Rajan.
Visa, re-skilling key challenges for IT industry: Eco Survey
The survey, tabled in Parliament today, said the Indian USD 150-billion IT-BPM industry is "feeling the pinch" of the global slowdown
India vulnerable to cyber crime, must upgrade defence: Study
Demonetisation and the subsequent push for digitisation has escalated risks relating to cyber crime and India needs to urgently upgrade its defences by setting up a cyber security commission on the lines of the Atomic Energy and Space Commissions, according to an IIT Kanpur study shared with Parliament's committee on finance.
'27,482 cyber security incidents reported in H1 2017'
Minister added that the types of cyber security incidents including phishing, scanning/probing, website intrusions and defacements, virus/malicious code, ransomware, denial of service attacks etc.
E-commerce companies at the receiving end, plaints swell to 49,000
For speedy redressal of these complaints, the National Consumer Helpline has partnered with 225 companies under a convergence programme, it said.
While Google and Uber fight, Indian govt throws out red carpet for self-driven cars
The move will allow Indian carmakers and technology firms to join the global race to develop self-driving cars by Google, Uber and others.
E-commerce companies demand exemption from GST
NASSCOM in its representation said the sector is creating huge job opportunities and allowing small industries to sell their products.
Internet users in India reached 342.65 mn in March
As per data shared by Telecom Minister Manoj Sinha in the Lok Sabha today, there were 34,26,54,750 internet subscribers as on March 31, 2016.


text:
Epic TV Series are gripping TV shows that chronicle grand stories of monarchs, rebels, armies, and apocalyptic events. The Epic has a long history in film, but only in recent years has the Epic come to television. And in a spectacular way. Beginning with the incomparable Game of Thrones, this is true Epic Theater.Read More
"Breaking Bad" and the REALLY Bad Boys of Television
The past couple of decades has produced some remarkably fascinating Bad Boy characters in television. These shows tend to have a single, not-so-nice male character whose actions determine every other character's lives and actions. Think about Tony Soprano. Sure, the show is called The Sopranos, but it's really about Tony and what he's cracking down on or who he's fighting with. Here's my survey of these shows and their lead characters.Read More
"The Sopranos" and Other Great TV Series About Crime and Criminals
The gangster, which is a form of anti-hero, is a well-established figure in storytelling for many centuries. Beginning with The Sopranos, these are TV series from the point of view of the criminal, and are some of the finest television shows ever made.Read More
"Sex and the City" + Other TV Series By & About Women
In the late 1990s a new kind of TV show emerged that was genuinely about women and women's lives, taking television's portrayal of women in new directions. In all of these shows, the lead characters were women, they had women friends, they talked to each other about their lives and problems, and the women characters were independent (even if married), strong, and making their own ways in the world. From "Sex and the City" onward, these are 5 TV shows for and about women.Read More
The Millennial's Guide to Gen-X Movie References In Stranger Things 2
The brilliant sci-fi series Stranger Things has returned to Netflix Streaming for its second season. As you watch it you might find yourself considering the cultural and generational motifs in the series...while you're getting your pants scared off. The great thing about throwback references? Many of these classics have a home at DVD.com so you can revisit them in all their glory.Read More


text:
Metro-East News
Davis disputes opponent Londrigan's ad about prescription drug prices. Who is right?
As the Nov. 6 election draws closer, a television ad being run in the northern part of the 13th Congressional District, which is expected to air in the St. Louis-media market, is already raising objections from one of the candidates.
The re-election campaign for U.S. Rep. Rodney Davis, R-Taylorville, is taking issue with a television ad discussing prescription drug prices.
The attack ad, which is narrated by an Edwardsville woman named "Sue," being paid for by the campaign of Betsy Dirksen Londrigan, the Democrat hoping to unseat Davis on Nov. 6. In the ad, Sue says "Davis voted to keep prescription prices high" and cites a Belleville News-Democrat article.
The May 4, 2017, article discusses how Davis, along with other metro-east congressmen, voted for the American Health Care Act, which was the House Republican effort to repeal and replace the Affordable Care Act, commonly referred to as Obamacare. The U.S. Senate never passed a bill to repeal the ACA.
The BND article doesn't discuss prescription drug prices. However, the Londrigan campaign in a subsequent interview, followed up and cited an NBC News article that says the AHCA did not address prescription drug prices.
Davis' camp counters that AHCA addressed the Medicare Part D doughnut hole.
Under the ACA, the Medicare Part D prescription drug doughnut hole would gradually close by 2020 in order to help seniors pay for prescription drugs. However, under the federal budget plan adopted this year, the doughnut hole is expected to close by 2019.
According to Kaiser Family Foundation, the AHCA didn't address the Part D doughnut hole, but kept the planned changes from Obamacare in place.
During an interview, Davis said the House has passed the "Know the Lowest Price Act," a bill that ends the gag clause for pharmacists, where pharmacists weren't allowed to say where customers could save on prescription drugs. The legislation is headed to President Trump for his consideration.
"That's clearly not in the best interests of drug manufacturers," Davis said.
Davis added the Centers for Medicare & Medicaid Services has announced Medicare part D premiums would drop because of more competition in the marketplace, and there that the FDA has streamlined the generic approval process, including a generic epinephrine pen coming out.
"We're going to continue to tell the truth, and we're going to continue to tell the truth when we believe that the facts are on our side that our opponent Betsy Londrigan is once again being dishonest," Davis said.
The Davis campaign said it expects the ad to start running in the St. Louis market.
Ashley Phelps, a Davis spokeswoman, said the Londrigan pattern with television ads is they start in the northern part of the Illinois 13th, and then work their way south into the St. Louis television market.
Londrigan's campaign would not comment on when it planned to start running the ad in the St. Louis market.
Disagreements over TV ads have already occurred in the race.
The Londrigan campaign has issued a cease and desist letter to television stations running ads paid for by the Congressional Leadership Fund, a super PAC that backs Republican candidates. The ads say Londrigan would support a "radical healthcare plan" that is too expensive.
"It could end employer provided insurance and end Medicare as we know it," the ad says.
The campaign wanted TV stations to stop running the ads.
"This advertisement is false, misleading and deceptive," the campaign's cease and desist letter said.
In a BND candidate questionnaire, Londrigan said she believes in opening up Medicare as a public option that would provide a competitor in the marketplace to bring costs down.
"Additionally, we must address the cost of prescription drugs," Londrigan wrote. "We need to allow Medicare to negotiate for prescription drug costs effective immediately."
The last several weeks between Davis and Londrigan is only expected to get more heated as at least one poll shows the race is tight.
Londrigan's campaign released a poll this week that showed Davis leading by one point: 49 percent to 48 percent. However, a Congressional Leadership Fund poll has the race at 50 percent to 37 percent. Londrigan's poll was in the field a few days after the CLF poll.
Sean Savett, a spokesman for the Democratic Congressional Campaign Committee, questions the make up of respondents in the CLF poll and says the CLF leans conservative.
As a sign of how close the race is, Vice President Mike Pence is scheduled to headline a fundraiser for Davis on Friday, which the Londrigan campaign has called a "rescue mission."
"D.C. Republicans are so worried about losing Rodney Davis' loyal vote that they're bringing in Mike Pence to raise even more money from the large corporations and special interests who he so clearly represents in Congress," said Emma Brown, Londrigan's campaign manager.
Davis said he has been preparing for a tough campaign, but is confident he will win re-election even as the Londrigan poll is showing the race is close.
"It's a message we now send to the folks that have elected and re-elected us before that we always said the race is going to be close and I sure hope no one takes any race for granted especially ours," Davis said. "We see different numbers, but having my opponent come out with something like that is good because it's a good reminder to a lot of the folks that are supporting us we can't take our foot off the gas. I clearly believe we'll be victorious on Election Day."


text:
Awesome Wood Fence Designs
Ideas we. Check out the awesome and water over the flower stencil before applying the most outstanding designs arts and get great home including wood rail fences designs for awesome gate repined on toby designs arts and save ideas so instead of art. Image of awesome wood fence designs, spectacular backyard for backyard fence can be just look at. Pueblo home kitchen traditional with wood and excellent fence options to help give you to get ideas and pools spectacular backyard deck designs spectacular backyard its high time as much time as possible outdoors in the thing. Classy image of the awesome.Tags : image of awesome wood fence designs


text:
The list of "high value added" professions that give access to NHR status in Portugal has changed. Occupations such as psychologists, architects, designers or archaeologists have left the list, and others such as directors of hotels and restaurants as well as dentists have been added. This is a real upheaval for the NHR status. In fact, the Portuguese tax authorities carried out a "thorough examination" of the list of professional activities recognized as "high value added" and which gave the right to the status of non-habitual residents. According to the order issued on Tuesday, "Portugal's economic situation has changed considerably since the publication of this table of activities, with a significant transformation of the dynamics of job creation". Non-usual residents in the occupations listed here are entitled to special rates on the IRS. What is the new list of professions recognized as "high value added" in Portugal? What about professions that come out of NHR status? Lisbob, the expatriate assistant in Portugal, tells you all about the new NHR status.
Winners and losers for the new NHR status
The Portuguese tax authorities justify this change by the difficulties encountered by the country's employers in various sectors "to hire workers with different skills and qualifications profiles". The first list of professions with access to the NHR status, published in 2010, was based on the codes of economic activities, called CAE. The Finanças have now abandoned this model and now adopt "a model based on direct correspondence in the codes of the Portuguese Classification of Occupations (CPP)".
The Portuguese Ministry supervised by Mário Centeno states that this change allows "the most immediate clarification of the interpretative doubts about the scope and scope of each of the activities listed in the table, because for each occupation code, a job description is given which takes into account examples, as well as the professions included and excluded, and on the other hand to ensure a better accuracy in the statistical comparability at European and international level in the different fields in which this classification is applied ". Understand who can.
Bye bye architects, welcome to the pickers
The new list of professions with access to NHR status lists professional categories such as doctors, engineers, university professors or information and communication technology specialists. However, other professions, such as psychologists, architects, geologists or archaeologists, have simply disappeared.
New labor requirements include activities such as hotel, catering, trade and other services managers, farmers and skilled workers in the fields of agriculture and livestock farming, skilled loggers, fishing and hunting workers, or plant and machinery operators and assemblers.
The order adds that the first list published in 2010 was appropriate for the start of the NHR tax system and that, "once tested by practice, it should benefit from further improvements", as is happening now.
The new list is valid from 1 January 2020 and according to the assessment of the evolution of the economic situation of the country, it can be reviewed within three years.
Some details on the new NHR list
A person who currently benefits from NHR status for a profession not in the new list will retain his rights. On the other hand, a person who would have been refused NHR status before December 31, 2019 but whose profession would be included in the new list, will not be eligible for NHR status.
As a reminder, the non-habitual resident status was created in Portugal in 2009 to promote the growth of Portugal. This status allows you to benefit from favorable rates for taxation on income, or even a total exemption in some cases.
New list of recognized "high value added" professions for NHR status
Here is the official list of professions recognized as "high value added" and entitled to NHR status as from 1 January 2020, published by the Portuguese Government.
"List of high value-added activities within the meaning of Articles 72 (10) and 81 (5) of the IRS Code"
I - Professional activities (CPR codes) :
112 - Director General and Executive Director of companies ;
12 - Directors of Administrative and Commercial Services ;
13 - Directors of production and specialized services ;
14 - Director of hotel, restaurant, business and other services directors ;
21 - Specialists in the physical sciences, mathematics, engineering and related techniques ;
221 - Doctors ;
2261 - Dentists and stomatologists ;
231 - University and higher education teacher ;
25 - Experts in information and communication technologies (ICT) ;
264 - Authors, journalists and linguists ;
265 - Creative Arts and Entertainment Artists ;
31 - Technicians and Intermediate Occupations in Science and Engineering ;
35 - Information and Communication Technology Technicians ;
61 - Farmers and skilled workers in agriculture and livestock breeding for trade ;
62 - Skilled workers in forestry, fishing and hunting ;
7 - Skilled workers in industry, construction and crafts, including in particular metallurgy, metalworking, food processing, woodworking, clothing, crafts, printing, precision instrument manufacturing, jewelers, craftsmen, electrical and electronic workers ;
8 - Plant and machine operators and assembly workers, namely operators of stationary machines ;
Workers in the above mentioned professional activities must have at least the level of qualification of the European Qualifications Framework Level 4 or the International Type Classification of Education of level 35 or five years of duly justified professional experience.
II - Other professional activities :
Directors of companies promoting productive investments, as long as they are eligible for eligible projects and tax concession agreements concluded under the Investment Tax Code, approved by Legislative Decree No. 162 / 2014 of October 31 ».


text:
Brokeback Mountain on the New Yorker website
Annie Proulx's prologue (that wasn't in the New Yorker)
Anne's Foolscap and Spreadsheet
Anne's Beat Breakdown
Anne's Crosswalk Document
Anne Hawley is the author of Restraint, a love story set in 19th Century London. She's a Story Grid Certified Editor specializing in literary and historical fiction, and is the producer of the Story Grid Editor Roundtable podcast.
[0:00:00.5] AH: Hello and welcome to the Story Grid Masterwork Experiment. Before we get started today, I want to remind listeners about Story Grid Live happening September 13th and 14th, 2019 in Nashville, Tennessee.
Story Grid has grown into a movement, followed by tens of thousands of writers from all over the globe who are serious about their craft. This live in-person weekend event will be full of information, inspiration and expertise, along with some food, fun and nerdery with your fellow Story Griders. Story Grid Live is the place to be for writers looking to deepen and grow their expertise in the craft of storytelling.
Step out of your routine to spend two days alongside other writers and storytellers like you. This is a chance not only to learn, but to connect with other amazing writers. Shawn Coyne and Tim Grahl will be presenting, along with special guest, Steven Pressfield. I'll be there and I hope you will too. Find out more at storygrid.com/live. That's storygrid.com/live.
Now on with our regular introduction.
My name is Anne Hawley and I'm an experienced novelist trying to ground my craft more solidly in Story Grid methodology. It's working. I've agreed to be the lab rat in the masterwork experiment, which Shawn Coyne is conducting as a test of his latest writing and editing methods. In the masterwork experiment, Shawn and I have analyzed Annie Proulx's novella, Brokeback Mountain, down to the beat level. I've taken on the challenge of constructing a new story using the same beats, but set in the England of Jane Austen, Lord Byron and Beau Brummell.
Last week, I established a new point of view narrative device and it gave me the momentum I needed to really get writing. This week, I turned in a new prologue, as well as several new scenes, including that all-important first intimate contact moment that's required of every courtship love story.
My story is running long already and I don't think I can bring it in at anywhere near Annie Proulx's elegant, distilled 11,500 words. Shawn's response to this conundrum is an inspiring discussion about the importance to a writer of living in the process and not living for the outcome. Also, there's some talk of hobo signs.
Put on your white coat and safety goggles and step into the laboratory for episode 9 of the masterwork experiment.
[00:02:28] AH: I just saw your request to edit, so I just granted you editing, or at least commenting on the document. I just now saw it, so I'm sorry I didn't get to that.
[00:02:37] SC: That's okay. It was just praise. You can live with that there, right?
[00:02:42] AH: Well, yeah. Oh. I got to tell you, this has been the most interesting process, Shawn. I am learning so much about what it means to write something. I tell you, I've had a number of people comment. I've gotten some nice feedback from people who have taken the time to e-mail me about the podcast. One of the things that people have said is I've mentioned that what I'm turning in is really rough. They go, "Ha, ha, ha. Really rough. It's beautiful," which is nice to hear. What they're talking about is my line writing is good. I have spent 57 years honing the language, right? The roughness is not in the line-by-line prose. The roughness is in the story structure.
The difference in perception about that is just amazing to me. I have never – even with four years of working with Story Grid, I have never quite gotten it into my body, right? The difference between being inspired and turning out beautiful pretty sentences and what a rough draft, first draft really is. It's like shoveling sand into the sandbox. I mean, it's just a big box full of sand. I haven't done anything with it yet.
[00:04:01] SC: Well, yeah. I mean, I have to say that I don't want to jinx us. In terms of an experiment, to get this early response, I just find myself reading your work. Even though I know where it's going, because we've so fully delineated all these beats, the discovery of your – that 57 years was not for naught, you know what I'm saying?
[00:04:31] AH: God, I hope not.
[00:04:32] SC: No, no. Absolutely not. That was one of the reasons that attracted me to you was in just the facility that you have, understanding what your voice is really all about. You have a specific writing voice that you developed over that time that is an automatic for you. What you've been struggling with is the difficult place people associate with structure, function and just organizational stuff that make up stories, because what I was taught and I think probably what you were taught as I was matriculating through various levels of school was that writers write. The more you write, the closer you will get to an epiphany. Those epiphanies will add up over time, such that the structural functional organization of a story will become uniquely yours. As opposed to the idea that like a chair, there is a structural functional organization to stories too, and it can be learned through craft and then applied to everyone's particular genius.
This notion that you have to make it through this passage until you are touched by a mystical greatness that gives you these tools intuitively, as opposed to learning something explicitly, has been deeply ingrained in I think everyone who's ever taken an English class in their life.
[00:06:11] AH: It certainly wasn't ingrained in me.
[00:06:14] SC: Yeah. The difficult thing – you're a very intelligent person. Intellectually, you could grasp, "You know what? Maybe that is BS. I'm willing to change my attitude and to explore this Story Grid methodology structural stuff and see if it's useful to me." Over a period of four years you're like, "Yeah, yeah. This is working. Yeah, I'm not fully buying everything, but I'm going to keep going and we'll see, we'll see, we'll see." I think the experiment has actually made you procedurally demonstrate this new understanding very, very specifically.
Each of these beats that you analyzed yourself and you formalized yourself, and I will remind you and everyone else listening that all of the beats that you're following were the beats that you determine not what I determine, which I think was really important. That was something that came organically through this process, because I initially believe like, "Oh, I will be the professor and I will show her the beats and then she will execute as exactly as I say," right? Not that that wouldn't have worked, but it was better, right?
Once I saw your beats, I'm like, "These beats are better. Go with her beats, because she's locked in in a way that I never will be." I think just reading the latest material that you've sent me, it's a joy. It's a joy to see that inherent execution of structural details that are workman-like crafty stuff that aren't why you wanted to become a writer in the first place. Using that craft and adding to your line-by-line skill set, it's created something in my estimation a sum greater than its parts.
That's the goal of any editor is to help the writer apply their finest skill set and add a little bit more and a tweak more and maybe a little bit more clarity, so that that special sauce that only they have can actually come out in a way that has never come out before. I'm not going to presume that this is the greatest thing you've ever written, but I think you've gotten a lot further along in this story in the amount of time that we've worked on it, than perhaps you have on previous things that you've worked on.
[00:08:38] AH: Oh, absolutely. I will tell anyone who asks that I came to Story Grid in order to find a way to fix the novel that I had already written that I knew wasn't working. I used it for that and it worked very well and I fixed the novel. Since then, I haven't written anything. Using these techniques as the basis to actually start something and write something, going down to this level of looking at the master work and trying to copy it basically, I mean, that idea was so difficult for me to grasp, until we actually – I mean, sat down and started doing this and you hired me on to take on this experiment. I would not have done it otherwise.
I just couldn't bring myself to look at something that I love, like Brokeback Mountain at that level of detail, break it down and ruin it. It ruins it when you break it down like this, you see all of its inner workings and guts and stuff and it loses all of its enchantment. That is what happens, right? Then a new level of incredible admiration and inspiration comes out of looking that closely at the inner workings of something that you love. It's been really remarkable.
[00:09:49] SC: I was listening to one of those online professors that you can download. I mean, that's the great thing about YouTube is that if you're interested in any one particular thing, you can usually find an expert who has set up a camera and they filmed themselves giving a lecture. Unfortunately, I forget which lecturer was explaining this. I believe it was part of John Vervaeke's Awakening from the Meaning Crisis, which I highly recommend.
Anyway, they were talking about the difference between idolatry and iconography. I found this fascinating. An icon is someone that you look up to, but you see them as a human being, right? They had the same problems we all do, but you look at their work and you say, "Well, if that person was capable of creating such great work, then the possibility that I could is it's available. That's actually a possibility, because that's a flawed human being. I'm a flawed human being. That flawed human being created a work of art and maybe this flawed human being could too." That's an icon. You'll get why I'm telling you this in a second.
Now an idol, an I-D-O-L, an idol is something that's not transparent. It's an opaque thing that we can never really grasp on to and understand it in any real function. An idol would be something that would be a magical totem, some person who came down from earth and was different than you, or I. They saw the world in a unique way, they were touched by greatness, they had a "talent" that you would never be able to approach. Oftentimes, I think what happens is that we get seriously confused between idols and icons. I don't believe that there are really any genuine idols, so to speak. I don't want to get into a controversy. Some people have them, okay.
I think the people who are creating stories and the people who are creating art and the people are doing amazing things, they are icons. Icons are inherently human beings, which means that we ourselves can aspire to iconography. When we look at a masterwork, our tendency is to be like, "Oh, my God. There's just no way I'm going to go near this thing. That person who wrote this masterwork has something that I will never have."
What I love about the masterwork experiment and this global masterwork gestalt that's part of the Story Grid methodology is that it converts, it looks at works as icons of virtue, as opposed to idols of we will never reach that arena. I wholly believe that if you don't set your sights at astronomically incredibly difficult places, you'll never even get near them. I don't think it's [inaudible 00:12:56] to say, "You know what? Let me take a look at this masterwork. Let me really analyze it beat by beat and see if I can't model my story on that." That's actually I think a tribute to the work, as opposed to a copy/exploitation.
Because you hold your story up to Annie Proulx's and they're completely different things. They're different characters, different time periods. Thematically and structurally, they share common things, but so does the Hobbit and Harry Potter, right?
[00:13:28] AH: Right.
[00:13:30] SC: I don't think we're committing blasphemy by really looking at the craft behind the masterworks. In fact, I think most people who write master works would be like, "Go to it, man." Sometimes I don't even know how I put it together. If you can tell me – I mean, Steve Pressfield always says that to me like, "Dude, I always love it when you analyze my stuff, because I had no idea a lot of the stuff I was doing."
When I look at it I go, "Oh, my God. I didn't know I was that smart." I think it's important to think about icons and idols and to steer away from idolizing human beings, to a place where you believe that you are so not near their skill set. Because it only holds you back and it will only serve as an excuse for you to not work.
[00:14:20] AH: Right. Yeah, I have turned out more words in the last week of actual fiction writing than in the last couple of years really.
[00:14:29] SC: Well, keep at it is all I can say, because you're working through this in a way that is exactly how I envisioned and hoped. I think all those worries that you had a few weeks ago, or a week ago about this thing not having life inside of it were completely unwarranted. What you have done here is one of the things I always like to say to somebody is everyone knows when a story is working. The reason why we know intuitively, and you don't have to be a Story Grid certified editor to know when a story is working. All you need to know is is the movie, or the film of the story clicking through your mind as you're reading it? Are you imagining the scenarios and the cloppings of the horses and the rain coming down and the smell of the woolen blankets as they're wet and the way the horses are neighing and afraid of the river. Is that running through your mind, or does it feel it's a chore to start to picture it?
That's testimony to not your structural knowledge. Your structural knowledge is inherent in you following the beats. That's all you have to do. What its testimony to is that 57 years of blue-collar work that you did to paint pictures with words, so that someone like me can start reading it and immediately, the film starts to run in my mind. That is just testimony to your hard work and your love of language and your love of research and your love of a time period. You're attracted to this very specific time period. From what I can see is that you have followed that interest, and so you have researched that time period. Yeah, you don't have all the details yet, but you know, you've pictured in your mind over and over and over again what it must have been like to have lived at that time.
All of that fun and fantasy imagining in your mind is really – you're taking dictation and you're applying it to the page with words that you've learned how to manipulate. I don't have any real commentary about the work that you sent me, other than to pick out some things that I was just – my heart melted when I read them, because I'm like, "Oh, my gosh. She gets it. She's doing it."
[00:17:03] AH: Well, I want to hear about those things.
[00:17:04] SC: Okay. Okay. Let me let me pull it up here. From the very start, the opening line and of course, you may or may not change this or whatever. What I loved is at the very first sentence, you say, "Josiah Reed drags himself from his palette in the loft of Salt Tire." I don't even know how to pronounce that.
[00:17:26] AH: I don't know either. I just picked it out of that.
[00:17:28] SC: "Saltire Manors, vast stables and climbs down the ladder." Okay, what I love about that very first sentence didn't really register to me until I'd reached about midpoint of the work that you had sent me. Because what you had done in that very first sentence is very similar to what Annie Proulx does all the time; is she's setting up the exposition that explains where he is in that very moment later on in the story. What I mean by that is that later on in the story, Mattie and Josiah have a conversation. It's the, hey, what school did you go to, conversation, right? When you first meet someone, you're getting acquainted. Like, "Well, I'm from Pittsburgh." "Oh, I'm from Portland." Oh, cool, two P towns, right? Youare hashing it back and forth.
He gives up a little bit about himself and he goes, "Oh, man. I grew up in a stable. Now, things are starting to turn around for me, but I really had humble origins. I was a foundling. I grew up in this hayloft." It's really terrific, because it tumbles all the way back to your very first sentence. Now this very first sentence, you're presenting a man at the later years of his life. Where is he? He's back where he started, isn't he?
[00:18:52] AH: Yeah, yeah.
[00:18:52] SC: He's come full circle. Just by that technique that you used that was inspired by Annie Proulx, you've successfully implied the nature of a heroic journey, so to speak, with the entire story just from that introduction. When a man's living in a hayloft, when he's older, we want to know, "Oh, my God. Well, how did he get there, right? What happened to this guy?" It's a great expositional thing that's loaded. It's loaded with so much ammunition to generate narrative drive that I absolutely loved it.
The other thing I loved about this new prologue is that our initial reaction about this guy is boy, he's got to be bitter, right? This is someone who's got to be really not so pleased that they're living in a hayloft. Instead, you play with it and this guy is feeding the birds, right? You've got Lord Black-feather, who he's probably – he feeds him every day, he sees him every day and gets a little – some corn and that's part of his life, right? It's a beautiful life. He has a relationship to the natural world in a way that any of us would say, "Hey, that's pretty cool." Bird comes by every day. He has a relationship. He throws down a little corn, the bird eats. This guy is really important in the universe, even though he lives in a hayloft. If he's not there to throw down that corn, that bird is going to miss them. That's great.
Then lastly, one of the really difficult things that you had to solve was what's the equivalent of the two shirts? What is the thing this guy – what's his totem? What's his temple? What's the thing that Ennis Del Mar remembers Jack? He's got his little shrine there. He's got his two shirts, he's got his picture of Brokeback Mountain, they're on the nail, he touches that shirt, he looks at that picture and he can get through another day. You had to solve that problem, but you couldn't use shirts, right? You couldn't cheat.
[00:21:01] AH: More postcards. Yeah.
[00:21:03] SC: More postcards, right? You can use a letter. This is a barn guy, right? This guy lives his life in a barn. He lives with horses. He lives with hay. What this reminded me of is there's this great moment ,the solution that you came up with which I'll share in a second. It reminded me of this great episode in Mad Men, that series about 1960s advertising. The episode was called The Hobo Code. In The Hobo Code, there is this moment where it's a flashback of the advertising executive, a guy named Don Draper in his childhood. He grew up on this falling apart farm somewhere in Indiana, or Illinois, or somewhere.
It's during the Great Depression, and these hobos would walk by and they would offer to do some menial labor to sleep in the barn and maybe get a meal. The hobos as they would travel along the road, they would bring a little piece of chalk with them. On the gate that led up to these houses, they would put little messages for each other, so that when they would see these messages, they would know whether or not the people in there were friendly, or if they weren't, or if they would run them off or whatever. The whole episode is based upon this hobo code, that this hobo, a traveling person who has no work puts on the gate of Don Draper's family's barn after his experiences there.
What you did to solve this problem reminded me of that beautiful decision in that television series, because what you've decided is to make a couple of notches in the side of the wood in the entry to the barn. Josiah, every time he walks in and out of that barn, he puts his two fingers and he rubs those two notches. That was beautiful to me, because it's a very tiny little thing in the side at the beginning of your prologue. Again, it's a setup. You pay off that setup far earlier than Annie Proulx did, which is testimony to you're going with Anne's flow, not Annie Proulx's flow.
The way it works in your story is that those two notches represent these two guys as they're getting to know each other. They're having this friendly debate about who's taller. What do men do? They got to get the data, right? They take the knife and they make the marks of who's taller. That I loved, because I immediately got it. Those two marks are what gets this guy through the door every single day. If those two marks weren't there, he would live in despair.
Before we even know this is a love story, right? Because you're not really pinpointing this is a love story at the beginning of the story. It's this fun, upstairs, downstairs Downton Abbey maybe thing. Those two things are part of the revelatory payoff. Oh, my God. This is a love story, that we find later on.
What's the other thing that I found? Well, the other thing that I just loved at the very end of your work was like Annie Proulx, the sex is almost incidental. It's two guys who find each other attractive at the end of the chunk that you sent me. Yeah, they get together, so what? You're not afraid of saying, "They're naked in front of each other." That image does flash in the reader's mind, but not in a gratuitous, or pornographic way, just natural to human beings. Then you cut. Like, enough. That's it. We're not here for that. We're here for something much deeper, much more meaningful.
Geez, Anne. I don't know what to say, other than keep going. Do you have any questions for me?
[00:25:06] AH: I do. There's a back and forth working process here that I'm – it's new to me, because I have this list of beats, right, that I'm supposed to be meeting. When I'm writing, like working on a scene, I've got to get the first sexual contact scene; I'm going to write it. Then I realize, "Oh, I should go back and look at my list of beats and see if I've missed one." I have. I missed a bunch of them. I know that there's no real strong exact correlation anymore on the crosswalk between this beat happens at this point in Brokeback Mountain and it hasn't happened yet in my story. That type of back-and-forth stuff. My sense is I should just keep going and then go back and see if I need to line up more beats, or – but what do you think?
[00:25:51] SC: Okay, that's a great question. I had a call earlier with our mutual friend, Valerie, about this very thing. Generally, here's what I think the beats are for; the beats are for inspiration. Therefore, clear directives to prime the pump of your imagination. Okay, so should you follow these beats religiously? My answer would be, if you don't need to, then don't.
The key thing about the beats is what they're saying to you are the moments in the story that you'll need to have in your story, whether or not they come at the specific time that Annie Proul put them in is not important. What's important is that these patterns, these beautiful patterns that Annie Proulx created in Brokeback Mountain, you want to have those patterns in your story too. Whether or not you're following the structural organization of those patterns to the tee isn't the important thing. The important thing is at the end of your draft, yeah, you're going to want to go back and go, "Did I hit that pattern? Did I nail that little beat? Is that in there somewhere? Have I put in the bit about there's a third party, sort of that character who catches them in the act? Joe Aguirre. Do I have a Joe Aguirre figure? Is it clear that he's probably holding this information for purposes of his own, that will later screw something up, or hurt these guys, or something?" That's what you want to look for. You have a really nice moment, a bunch of moments in there where you're like, "The gun guy."
[00:27:42] AH: You're right. The gun guy, whose name I haven't figured out yet.
[00:27:44] SC: That's great. The gun guy. That's all you need to know. There's the guy who takes care of the guns, the guy who takes care of the logistical details, that's Mattie, and there's the guy who takes care of the horses, that's Josiah, right? That's what you need. You need the gun guy, the logistical guy and the horse guy, all to get this package from here to there. That's the other great thing is that you put them on the road, which mirrors all of our movements through the universe.
They're on the road and you've got this gun guy who's this wild card. You're not really sure how to use him yet, but you're not letting that stop you writing. You're setting up possible all kinds of things. If you did not have the gun guy, then you lose the triangle of relationship exponential possibilities.
In a one-on-one relationship, one person can like one person, the other person could like the other person, they could both like each other. I'm talking very generally and generically. When you have a triangle, it adds so much more nuance. Person one could like person two. Person two could like person three. Person three could like person one. Person one could then like – right? You can really fiddle around with the relationships when you have three in the pot, as opposed to two.
That's why you always have the love triangle, because it adds complications and all kinds of nuance to a story that could get really, "I love you. I love you not. I love you. I love you not." That just doesn't really go very far. The gun guy is a great idea. Just to get back to your central question, no. Go as directed and then later on, what you're going to do is you're going to just review, review what Annie Proulx did. Say she did this, this, that and this. Okay, did I do that? Yeah, it's here. Yeah, this one's over here. That's okay, because it works. Does that work? Yeah, that works. I missed that one. Is it crucial? I don't think it's crucial, so I'm going to I'm going to give myself the liberty not to do that beat.
You see, that's where your editor comes in. Your editor head is going to be far better at telling you whether or not a beat that you may have missed should be in or not be in. You might say, "You know what? I'm forcing this. I don't need it." While you're doing your writing, I always say this, just keep that writer's hat on. Keep momentum. Keep the TKs. Use TKs until you – to your heart's content. Gun guy TK is great. Keep doing it.
[00:30:23] AH: TK village. TK name of in. I got a lot of TKs in here. I will admit, I did spend a little bit of time with maps and street view, trying to get a feel for the scenery, but I'm trying not to get too deep into that.
[00:30:38] SC: Yeah, it's working.
[00:30:40] AH: I don't want to write myself into a corner that then historically, or geographically I can't write my way back out of if I find out that it's wrong. I'm trying to walk that line a little bit. I do have some concerns about the word count. I just can't see myself bringing this story in at 11,000 words.
[00:30:59] SC: Well, who cares? Don't worry about it.
[00:31:02] AH: Okay. That's all I need to hear, because this thing could become a full-length novel.
[00:31:07] SC: Well, yeah. If it works 16 – if it works at 16, great. I mean, one of the things that you have to remember about your story versus Annie Proulx's story is that the American West is a hardscrabble sensibility. It's a world in which the average American and even international readers who are familiar with the American West, there's a whole universe attached to it. It allowed her to use shorthand and not really have to get into the eloquence of the details of sunsets. I mean, she does it very well. She does it in one sentence, what most of us would take three sentences to do.
My only point is that you're writing a historical. Now hers was historical, but it was within the realm of modern-day life. It was the turn between – it was really the death of the Old West that she was talking about. You're taking us into a whole other area, which is going to require more description. It's going to require more stuff, more the taproom. I love the taproom. Who wouldn't love the taproom?
To not include fun taproom moments because of a word count would be a mistake, because you're not satisfying the core readership that's going to want to read this story. This story is a historical. What do readers of historical fiction usually want? They want that detail. They want that resolution. They want to know what bridle the guy uses, or whatever.
[00:32:48] AH: Right. A little bit more about horses, a little bit more about the clothes, things like that, or what historical readers read for.
[00:32:56] SC: Yeah. Never worry about going too long. You can really through levels of editing and line editing, you can – I'm in the middle of writing some back cover copy for some books, right? My first drafts are always about 500 words. I'm like, "God. Man, I can't put 500 words on the back cover of a book. I got to bring it down to 200." Then I'm like, "There's no way I can cut this." Then I let it sit overnight. Then I come in the next morning and I realize I have three sentences saying the same thing. Those are going and let me put that in there. That's what editing is for. If I didn't have those 500 words, I wouldn't have been editing anything. Yeah. Short answer is don't worry about word count.
[00:33:40] AH: Okay, good. Thank you. That relieves my mind. I have to say just for people listening who may know my other writing, writing this with the awareness of the brevity that Annie Proulx uses, it's not the American West, but this condensed, distilled way that she has writing. Trying to keep that in mind has been really interesting. I mean, it has stopped me from putting so many things on the page that I would have put on a page before. It's been a wonderful education in getting to better brevity in my work. Anything you want to bring up about the process?
[00:34:18] SC: Yeah. One of the things that people I think get confused about when they're writing something is they're results-oriented. What that means is that they're concentrating on the end point far too much. It's almost like they're fixated on it. The end point being something like, "Oh, I've got to do this in 10,000 words. Therefore, that's my result that I need to deliver." Or, "My second novel has to sell at least as well as my first novel. My first novel sold 10,000, so that my goal is I need the second novel to sell 11,000 and my first novels inciting incident was this inciting incident, so I better just make that inciting incident a little bit better, so that I can sell a little bit more number of copies."
That's like a results-oriented mindset. It's what I call the having mode. I think I've talked about Erich Fromm before, but he was a psychologist in the 1970s who clearly identified these two ways of looking at the world. It's we go after haves and we choose to be, right? Having is about getting that 11,000 copy sell for my second novel. If I could have that 11 copy sell for my second novel, then I will feel better.
Okay, now to be is more about, I am working on creating something and I'm not really sure where it's going to go, but I'm going to trust the process. I'm going to trust the things that I've done before, because they've brought me through the forest before and they will this time too. In fact, when I trust the process, I actually learned a whole bunch of new tricks. Those are processed-driven people and those are people who are living in the be mode. They're being writers who are trusting their process, which they have developed over say, 57 years.
That way of thinking, in my estimation I can only speak from personal experience, is far more satisfying and far more interesting a way to pursue the thing that you're most obsessed about, than it is to say, "I will feel better about myself if I get a bigger house. Therefore, if I have a bigger house, I will have more respect from other people, which will increase my self-esteem, which will make me feel better." That can be a very slippery slope. It's not to say that we don't need to attain things. Of course, we do. We need to be very clear about what our goal is. If I have to make breakfast, I make my breakfast, right? Having breakfast at the four seasons isn't so important to me.
When we are working on our projects and we're saying, "Well, I'm not at that 15,000-word level yet, or I'm not hitting my page count, or I'm not going – this doesn't seem to be that thing that's going to sell a lot of copies," it's a trap. Because we're living in a have mode when we're trying to apply our being in a process.
That's why I think the Story Grid experiment for you has been interesting, because we are so concentrated on the process, not the have at the end, right? I'm sure in your mind you're like, "Geez, I hope I can have this published in a way that will be interesting and that people who are e-mailing me about how much they're enjoying the story will be able to buy it. Then maybe I can get that new coffee table if it does well enough." Now that's perfectly reasonable, but it's not something that you should be focusing on when you're doing the creation.
That's what my magic wand, my magic thing that I would want for Story Grid is it's process-driven as opposed to results-driven. Many times in my career, it's all about results. Shawn, can you take this thing and turn it into a best-seller, right? If I do do that, it's satisfying, but not really, because it's all about that end-result. Guess what happens when I do take that thing in and it doesn't become a best-seller? Then people are upset. It's the same process that brought both results.
As long as I focus on the process of learning more about story and applying the global principles that I can call from examining multiple stories, then that's all I need. That's living in the process. It's been my experience that the more I live in the process, the less I have to worry about having. The haves almost start to take care of themselves. I think it's important for everyone listening that if you're listening to Story Grid Podcast, or you're listening to the Story Grid Editors Roundtable with the goal of learning some secret sauce that's going to get you what you want, as opposed to listening to enjoy the process of the examination, you're going to be disappointed. You're going to say, "Yeah, I wish Shawn would shut up and just tell me the secret sauce."
[00:39:48] AH: Hand me the magic key.
[00:39:49] SC: If he could boil everything down into seven paragraphs and really spit it out, so that I can make notes and then apply that formula to my stuff and then I can get a best-seller, that would be much more preferable than listening to him talk about process for the end. My point is the process is all that matters. It's the work. It's the work that frees us from worrying so much about whether or not somebody thinks we're cool or not. Because when we're doing the work, we find ourselves getting more and more insights, having more and more flow, enjoying the process more.
It doesn't mean that every time we do the process, we're going to get a good result. It just means that we're doing what we should be doing. That if we keep doing that thing and keep trying to challenge ourselves to get a little bit better at doing it, each and every time we do it, the probability is that eventually, eventually the results will be commercially successful and we can get our new coffee table.
[00:40:57] AH: That's a wrap for episode 9 of the masterwork experiment. Once again, we ran into a technical problem that cut us off a little short. In a follow-up e-mail, Shawn asked me to share that the goal for this episode was just to encourage me and all of us to keep our writing hat on, to trust the process and let it bring us to shore. Leave the results until it's time to edit.
In the show notes, you'll find links to all my working documents, John Vervaeke's lecture on Awakening from the Meaning Crisis, which Shawn referred to in this episode, and an article on hobo signs just for fun.
For everything Story Grid related, visit storygrid.com. If you'd like to learn more about me and my writing and editing work, you'll find me at annehawley.net. That's A-N-N-E-H-A-W-L-E-Y.net, where I'm posting my draft scenes complete with TKs and temporary character names, like gun guy. You can check all that out on my blog.
You'll also find me every Wednesday on the Editor Roundtable Podcast, where I team up with four of my fellow certified Story Grid editors to analyze the story structure of a movie, or a novel each week.
To support the podcast, tell other authors about us and leave us a rating and review. Join us again next week for the final episode of the masterwork experiment when I bang out all my remaining scenes and we discuss the editing steps that are going to have to take place between that point and eventual publication.
Thanks for listening. We'll see you next time.


text:
Back to Journals » OncoTargets and Therapy » Volume 11
Osteopontin promotes cancer cell drug resistance, invasion, and lactate production and is associated with poor outcome of patients with advanced non-small-cell lung cancer
Authors Ouyang XP, Huang Y, Jin X, Zhao W, Hu T, Wu F, Huang J
Received 28 January 2018
Accepted for publication 20 July 2018
Published 19 September 2018 Volume 2018:11 Pages 5933—5941
DOI https://doi.org/10.2147/OTT.S164007
Checked for plagiarism Yes
Review by Single-blind
Peer reviewers approved by Dr Narasimha Reddy Parine
Peer reviewer comments 3
Editor who approved publication: Dr Samir Farghaly
Xiaoping Ouyang,1,2 Yumin Huang,2 Xing Jin,3 Wei Zhao,4,5 Tao Hu,2 Feng Wu,2 Jianan Huang1
1Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Soochow University, Suzhou, People's Republic of China; 2Department of Respiratory Medicine, The Affiliated Hospital of Yangzhou University, Yangzhou, People's Republic of China; 3Department of Clinical Laboratory, The Affiliated Hospital of Yangzhou University, Yangzhou, People's Republic of China; 4Department of Clinical Biochemistry, School of Laboratory Medicine, Chengdu Medical College, Chengdu, People's Republic of China; 5Department of Pulmonary Medicine, Jinling Hospital, Nanjing University School of Medicine, Nanjing, People's Republic of China
Background: Osteopontin (OPN), a member of the small integrin binding ligand N-linked glycoprotein family, has been analyzed in numerous types of human malignancy.
Purpose: The present study detected the expression levels of OPN and evaluated its role in tumor progression in patients with non-small cell lung cancer (NSCLC).
Patients and methods: OPN expression levels were detected using immunohistochemistry in 101 NSCLC tumors. The mRNA and protein levels have significant difference between advanced NSCLC and stage I/II NSCLC. The drug resistance, invasive ability and lactate production of NSCLC cancer cell lines (A549 and SK-MES-1) were detected in cancer cells with the disturbance of OPN.
Results: Immunostaining indicated that OPN was primarily expressed in the cytoplasm of NSCLC cells. Moreover, OPN correlates with NSCLC clinical traits. The results demonstrated that OPN expression levels significantly correlated with cancer differentiation, distant metastasis and the efficacy of platinum-based treatment. Notably, the results identified OPN expression levels as a potential factor for predicting the response of cells to first-line platinum-based chemotherapy using multivariate analysis, as well as predicting cancer differentiation and distant metastasis. Additionally, the abrogation of OPN levels reduced lactate production in NSCLC cells and occurred along side with the downregulation of lactate dehydrogenase A (LDHA).
Conclusion: The results of the current study suggest that OPN may be able to predict poor prognosis and cisplatin resistance in patients.
Keywords: osteopontin, non-small-cell lung cancer, clinical outcome, drug resistance, invasion, lactate production
Lung cancer is the most frequent cause of cancer-associated mortality worldwide.1,2 Non-small-cell lung cancer (NSCLC) accounts for ~85% of lung cancer cases and has an overall 5-year survival rate of <20.0%.1 This low rate may occur as a result of the majority of cases being diagnosed as late-stage cancer and therefore being unsuitable for curative surgery.3
A number of clinical trials have suggested that adjuvant chemotherapy is the optimal standard treatment for patients with NSCLC at present.1,4,5 However, 70%–80% of patients who receive cisplatin-based chemotherapy develop drug resistance.6 Therefore, predicting tumor drug resistance using novel prognostic biomarkers is important for the treatment of patients with NSCLC. Novel biomarkers may be useful to stratify patients with NSCLC in order to select patients who may benefit from receiving aggressive adjuvant chemotherapy.
Osteopontin (OPN) is expressed by macrophages, fibroblasts, and lymphocytes, as well as endothelial, smooth muscle, and epithelial cells. It is involved in multiple physiological processes including cell adhesion, wound healing, bone resorption, angiogenesis, immune system responses, and tissue remodeling.7,8 OPN is overexpressed in numerous types of cancer including breast, lung, colorectal, stomach, and ovarian cancer as well as melanoma.9,10 Proteomic analysis of tumor tissue has identified OPN as an effective biomarker for numerous types of cancer and a number of studies on clinical specimens have correlated OPN expression levels with high-grade malignancies.7,8,11,12 High levels of OPN expression in lung cancer positively correlate with reduced patient survival rate.13 Additionally, highly expressed OPN in tumor cells is reported to be associated with a poor survival rate of patients with stage I14,15 and aggressive16,17 NSCLC. OPN levels in the circulating plasma from 158 patients with NSCLC were significantly higher compared with patients with benign pulmonary disease and 25 healthy control groups.17 Taken together, previous reports suggest that OPN may aid multiple pathological processes involved in NSCLC, including cancer cell proliferation, invasion, tumor progression, and metastasis. However, the correlation between OPN expression levels and chemotherapeutic efficacy in patients with NSCLC remains to be elucidated.
A key checkpoint of anaerobic glycolysis is the conversion of pyruvate to lactate and this is catalyzed by lactate dehydrogenase A (LDHA).18 Numerous types of cancer exhibit elevated LDHA and this has been associated with tumor cell growth, survival, and invasion.19–21 In a previous study, high levels of LDHA expression were identified to be correlated with drug resistance and a decreased survival time in numerous types of myeloma.22 It remains to be elucidated whether there is a correlation between OPN and LDHA expression levels.
The aim of the present study was to assess OPN expression levels in 101 consecutive patients with NSCLC and correlate its expression levels with the response to platinum-based chemotherapy and clinicopathological parameters. The current study also examined the effect of OPN expression levels on NSCLC cell drug resistance, invasion, and lactate production.
Materials and methods
NSCLC patients and tissue samples
Between March 2010 and August 2016, a total of 73 patients with advanced NSCLC treated with chemotherapy, 28 patients with stage I and II NSCLC, from the Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Soochow University (Suzhou, China) were selected to participate in the current study. The definition of NSCLC stage was formulated following reference instructions.23 Patients were diagnosed with NSCLC following a bronchoscopic lung biopsy and advanced-stage NSCLC included patients with stage III and IV NSCLC that were considered inoperable. Patients underwent imaging studies prior to treatment and received first-line chemotherapy regimens with platinum-based reagents. Treatment response evaluation was performed following the fourth cycle of chemotherapy. The present study was approved by the Institutional Ethics Committee of the First Affiliated Hospital of Soochow University. Written informed consent was obtained from all patients.
Immunohistochemistry (IHC)
Paraffin tissue sections were formalin fixed in preparation for IHC. The tissues were cut into 4-μm sections, dewaxed, and rehydrated and, to inactivate the endogenous peroxidase, the tissues were incubated in 0.3% (v/v) hydrogen peroxide and 0.01 M PBS (pH 7.2) for 20 minutes. Antigen retrieval was performed under high pressure for 2 minutes using 0.01 M sodium citrate buffer (pH 6.0). The sections were immunostained with 2 μg/mL anti-OPN primary antibody (Bioworld Technology Co, Ltd, Nanjing, China) overnight at 4°C and stained with a horseradish peroxidase (HRP)/fragment antigen binding polymer-conjugated secondary antibody (OriGene Technologies, Rockville, MD, USA) for 30 minutes at room temperature. Finally, diaminobenzidine was used to activate the HRP-conjugated secondary antibody at room temperature for 1 minute and the tissue was counterstained for 15 minutes with hematoxylin. At least five fields were randomly selected for each section, which were examined and scored by two independent investigators with no knowledge of the clinical details of the patients. The immunostaining was scored as previously described.24 A score of high expression was assigned when ≥30% of the cancer cells were positive for OPN expression and low expression was scored when <30% of the cells were negative for OPN expression. This scoring method was used, as it was preferred to multiple cutoffs that were examined (0%, 20%, 50%, and 100%). The subcellular localization of the staining was also evaluated.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA from the cells was extracted with Trizol® (Thermo Fisher Scientific, Inc, Waltham, MA, USA). The expression levels of hmgb3 were detected using RT-qPCR. The primers used were as follows: sense, 5′-GCCACAGCATCTGGGTATTT-3′; antisense, 5′-GTGATTTGCTTTTGCCTCCT-3′. β-Actin was used as an internal control and the primers for β-actin were: sense, 5′-AGCGAGCATCCCCCAAAGTT-3′; antisense, 5′-GGGCACGAAGGCTCATCATT-3′. RT-qPCR was performed using the SYBR® Green (Takara Biotechnology Co, Ltd, Japan) dye detection method on an Applied Biosystems StepOne™ Sequence Detection System (Thermo Fisher Scientific, Inc).
Cell lines, small interfering RNAs (siRNAs), and OPN-overexpressed plasmids transfection
A549, SK-MES-1, and H1703 human NSCLC cell lines and a normal lung bronchial epithelial cell line 16HBE were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in an atmosphere of 5% CO2 and 95% air. The siRNAs for opn (siOPN-1: 5′-guGAUUGAUAGUCAGGAACUUuc-3′; siOPN-2: 5′-guGAACGACUCUGAUGAUGUAUUuc-3′) and the negative control siRNA (siNC: 5′-guACUCUAUCUGCACGCUGACuc-3′) were synthesized by Guangzhou RiboBio Co, Ltd (Guangzhou, China). The full-length OPN sequence was synthesized and subcloned into pcDNA3.1 vector (Thermo Fisher Scientific, Inc). The plasmids or siRNAs were then transfected into cells separately using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Inc). Transfection was performed as previously described.25
Western blot analysis
A total of 20 μg of protein was electrophoresed on a 10% SDS-PAGE gel, transferred onto polyvinylidene difluoride membrane, blocked, and incubated with primary antibodies (OPN; Bioworld Technology Co, Ltd; 1:1,000 dilution; LDHA; Bioworld Technology Co, Ltd; 1:5,000 dilution). β-Actin (Bioworld Technology Co, Ltd; 1:1,000 dilution) was used as a loading control. The membrane was incubated with a HRP-conjugated secondary antibody at room temperature for 2 hours. Protein bands were detected on a G:Box Chemi-Imager (Syngene, Frederick, MD, USA) using a SuperSignal West Pico Chemiluminescent Detection System (Thermo Fisher Scientific, Inc).
MTT assay
A549, SK-MES-1, and H1703 cells were plated at a density of 1×103 cells/well in 96-well plates in 100 μL medium. Cells were transfected with plasmids or siRNAs for 24 hours prior to cisplatin (CDDP; Sigma-Aldrich Co, St Louis, MO, USA; doses of 0, 0.5, 1, 5, 10, or 50 μg/mL), lactate (Sigma-Aldrich Co), or sodium oxamate (Sigma-Aldrich Co) being added to the medium. At the end of the test, 10 μL MTT was added to each well and the mixture was incubated at 37°C for 4 hours in the dark. The media and MTT were removed and 100 μL dimethyl sulfoxide was added to each well to dissolve the formazan crystals. The absorption value was recorded at a wavelength of 490 nm.
Transwell invasion assay
A transwell invasion assay was performed using a 24-well transwell chamber with a pore size of 8 mm (Costar®; Corning Incorporated, Corning, NY, USA). NSCLC cells (2×103) were transfected with siOPN-1/siOPN-2 or siNC and were seeded in serum-free medium into the insert chambers, which were coated with Matrigel (BD Biosciences, San Jose, CA, USA). Medium supplemented with 10% fetal bovine serum was added to the lower chamber as a chemoattractant. Following incubation for 24 hours, the invaded cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and quantified from five microscopic fields.
Lactate production measurement
NSCLC cells were transfected with siOPN-1/siOPN-2 or siNC for 48 hours and were seeded (1×105 cells/well) into 35-mm dishes and incubated at 37°C. Following incubation for 36 hours, the cells were separated using centrifugation. Lactate production levels in the culture media were detected using the Lactate Assay Kit (BioVision, Inc, Milpitas, CA, USA) according to the manufacturer's protocol.
Statistical analysis
Statistical analyses were performed using SPSS version 16.0 (SPSS Inc, Chicago, IL, USA). For continuous and categorical variables, a two-sample t-test for independent samples and Pearson chi-square test, respectively, were performed. One-way analysis of variance was performed to compare the means of two or more independent groups. P<0.05 was considered to indicate a statistically significant difference.
OPN is overexpressed in tissue from patients with advanced NSCLC
The expression levels of OPN were detected in tissue samples from 28 patients with stage I/II and 73 patients with advanced-stage NSCLC. OPN was predominantly detected in the cytoplasm (Figure 1A–C). Analysis of the IHC scores indicated that OPN was significantly upregulated in advanced NSCLC cases (P=0.0424; Figure 1D). RT-qPCR was performed to evaluate OPN expression levels in tissues collected from 22 patients with stage I/II and 31 patients with advanced-stage NSCLC. These results suggested that OPN expression was significantly upregulated in tissues from patients with advanced-stage NSCLC, compared with those from stage I/II NSCLC group (P=0.0246; Figure 1E).
OPN expression levels correlate with clinicopathological parameters in patients with advanced NSCLC
The correlation between OPN expression levels and the clinicopathological characteristics of patients with advanced NSCLC was analyzed (Table 1).
A total of 42 (57.53%) patients were placed in the high-OPN expression group, with the cytoplasm of >30% of tumor cells exhibiting positive staining. Table 1 demonstrates that OPN expression levels were significantly correlated with tumor differentiation (P=0.031), metastatic status (P=0.019), and response to platinum-based chemotherapy (P=0.038).
OPN expression levels are associated with the efficacy of platinum-based therapeutic agents in patients with advanced NSCLC
Table 1 indicates that OPN expression levels were significantly correlated with the patients' response to platinum-based therapeutic regimens (P=0.038). Following corrections for age, gender, smoking history, pathological type, grade of metastasis, and differentiation, logistic regression analysis indicated that OPN expression levels (P=0.005), grade of metastasis (P=0.021), and level of tumor cell differentiation (P=0.031) may be independent predictors of the tumor response to first-line platinum-based chemotherapy (Table 2).
Downregulation of OPN expression decreases NSCLC cell drug resistance and invasive ability
To evaluate the effect of OPN levels on NSCLC cell progression, the expression of OPN was firstly detected in NSCLC cell lines and immortalized human normal bronchial epithelial cells (16HBE). The protein level of OPN was higher in NSCLC cells compared with 16HBE (Figure 2A). Next, knockdown of OPN using RNA interference was performed on A549 and SK-MES-1 cell lines, and overexpressed in H1703 cells. The results revealed that overexpression of OPN significantly increased the proliferation of H1703 cells and decreased the sensitivity of cells to CDDP (Figure 2B). The decrease of OPN protein attenuated the proliferation of A549 and SK-MES-1 cells, and increased the sensitivity of NSCLC cancer cells to CDDP (Figure 2C and D). The MTT assay demonstrated that the CDDP IC50 values of siOPN-1/siOPN-2-transfected A549 and SK-MES-1 cells were 5.07/5.12 and 4.87/5.00 μg/mL, respectively, which was lower compared with the control cells (A549, 8.58 μg/mL; SK-MES-1, 7.56 μg/mL; Figure 2C and D). The transwell assay evaluated the cell invasion of NSCLC cells following siOPN or siNC treatment. Figure 2E and F reveals that A549 and SK-MES-1 cell lines exhibited decreased invasive abilities when OPN expression levels were abrogated using siOPN-1/siOPN-2.
Reduced OPN levels correlate with a decrease in LDHA expression levels and lactate production
To identify the underlying mechanisms of the effects of OPN, the cisplatin resistance of NSCLC cells, Gene Expression Omnibus repository DataSet Record GDS1650 was screened for correlation between OPN expression levels and drug resistance. Notably, as seen in Figure 3A, OPN mRNA levels significantly correlated with LDHA, which has been associated with the response to chemotherapy and tumor invasion.19,20 Additionally, the knockdown of OPN using siOPN-1/siOPN-2 decreased LDHA protein levels (Figure 3B) and lactate production (Figure 3C) in NSCLC cell lines compared with cells treated with siNC. Finally, to verify lactate contributes to OPN caused CDDP resistance in NSCLC cells, the lactate treatment (5 mM, 24 hours) was found to increase NSCLC cells CDDP (5 μg/mL, 24 hours) resistance and partial rescue OPN-deficiency led CDDP sensitivity (Figure 3D). This suggests that OPN levels may impact the efficacy of cisplatin-resistance ability of NSCLC cells through the regulation of LDHA.
OPN is a secreted chemokine that has been reported to exhibit overexpression in numerous types of cancer, including breast, lung, colorectal, stomach, and ovarian cancer as well as melanoma.9 In lung cancer, OPN is reported to be overexpressed in tumor tissues and correlates with poor clinical outcomes,13–15,26–28 and also in the plasma of patients with NSCLC.29 Cui et al30 demonstrated that the interaction between OPN and αvβ3 integrin may possess an important role in the tumor growth of human lung cancer cells when injected into mice. OPN may also promote the growth of metastatic tumors in a preclinical model of NSCLC.31 These previous studies suggest that OPN may serve as an important oncoprotein in the growth and progression of NSCLC.
In the present study, the expression levels of OPN were detected in NSCLC using IHC and RT-qPCR. Compared with stage I and II specimens, the advanced-stage NSCLC tissue exhibited an increased level of expression of OPN mRNA and protein. Therefore, it was hypothesized that OPN may contribute to NSCLC progression. Inconsistent with previous reports,32–34 we found no significant difference of OPN between stage I/II NSCLC tumors and adjacent normal lung tissues (data not shown); this may have been caused by the small sample size in our study (28 stage I/II NSCLC tumors vs 28 adjacent normal lung tissues). The analysis of OPN expression levels in 73 advanced NSCLC patients indicated that OPN overexpression was significantly associated with tumor differentiation (P=0.031), metastasis status (P=0.019), and the response to platinum-based chemotherapy (P=0.038). Combined with the reports of Shojaei et al and Li et al,35,36 OPN levels were correlated with the progression of NSCLC and the efficacy of platinum-based chemotherapy in patients with NSCLC. However, the underlying molecular phenomenon is rarely uncovered. Here, we emphasized on OPN-mediated NSCLC tumor invasion and CDDP resistance was found to be correlated with NSCLC cells, aerobic glycolysis (lactate production).
A metabolic alteration exhibited by cancer cells is the Warburg effect, which is associated with malignant progression and drug resistance.37 LDHA, a key enzyme in the glycolytic pathway, correlates with resistance to chemotherapy and cancer progression in multiple types of tumors.19 The current study indicates that OPN expression levels correlate with LDHA, affecting its protein levels and, therefore, lactate production. This suggests that the overexpression of OPN in NSCLC may promote platinum resistance and invasion through LDHA upregulation.
In conclusion, the increase in OPN expression levels was associated with poor outcome in patients with NSCLC in the current study. OPN may be a predictor of the tumor response to first-line platinum-based chemotherapy. The downregulation of OPN expression decreased the resistance of NSCLC cells to cisplatin and also reduced the invasive and lactate producing abilities of tumor cells. These results suggest that OPN expression levels may be used as a biomarker for patients with advanced NSCLC and a potential therapeutic target for increasing the efficacy of platinum in NSCLC tumors.
This work was supported by the National Natural Science Foundation of China (81602636) and Nanjing Medical Science and Technology Development Project (ZKX15049).
The authors report no conflicts of interest in this work.
Miller KD, Siegel RL, Lin CC, et al. Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin. 2016;66(4):271–289.
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7–30.
Goldstraw P, Ball D, Jett JR, et al. Non-small-cell lung cancer. Lancet. 2011;378(9804):1727–1740.
Cheong KA, Chrystal K, Harper PG. Adjuvant chemotherapy in non-small cell lung cancer. Int J Clin Pract. 2007;61(1):143–146.
Qi L, Li Y, Qin Y, et al. An individualised signature for predicting response with concordant survival benefit for lung adenocarcinoma patients receiving platinum-based chemotherapy. Br J Cancer. 2016;115(12):1513–1519.
Allingham-Hawkins D, Lea A, Levine S. ERCC1 expression analysis to guide therapy in non-small cell lung cancer. PLoS Curr. 2010;2:RRN1202.
Bandopadhyay M, Bulbule A, Butti R, et al. Osteopontin as a therapeutic target for cancer. Expert Opin Ther Targets. 2014;18(8):883–895.
Ahmed M, Behera R, Chakraborty G, et al. Osteopontin: a potentially important therapeutic target in cancer. Expert Opin Ther Targets. 2011;15(9):1113–1126.
Coppola D, Szabo M, Boulware D, et al. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin Cancer Res. 2004;10(1 Pt 1):184–190.
Bandopadhyay M, Bulbule A, Butti R, et al. Osteopontin as a therapeutic target for cancer. Expert Opin Ther Targets. 2014;18(8):883–895.
Fedarko NS, Jain A, Karadag A, van Eman MR, Fisher LW. Elevated serum bone sialoprotein and osteopontin in colon, breast, prostate, and lung cancer. Clin Cancer Res. 2001;7(12):4060–4066.
Weber GF, Lett GS, Haubein NC. Osteopontin is a marker for cancer aggressiveness and patient survival. Br J Cancer. 2010;103(6):861–869.
Chambers AF, Wilson SM, Kerkvliet N, O'Malley FP, Harris JF, Casson AG. Osteopontin expression in lung cancer. Lung Cancer. 1996;15(3):311–323.
Donati V, Boldrini L, Dell'Omodarme M, et al. Osteopontin expression and prognostic significance in non-small cell lung cancer. Clin Cancer Res. 2005;11(18):6459–6465.
Boldrini L, Donati V, Dell'Omodarme M, et al. Prognostic significance of osteopontin expression in early-stage non-small-cell lung cancer. Br J Cancer. 2005;93(4):453–457.
Yu TT, Han ZG, Shan L, et al. Expression of osteopontin in non-small cell lung cancer and correlative relation with microvascular density. Asian Pac J Cancer Prev. 2014;15(1):29–32.
Hu Z, Lin D, Yuan J, et al. Overexpression of osteopontin is associated with more aggressive phenotypes in human non-small cell lung cancer. Clin Cancer Res. 2005;11(13):4646–4652.
Nair VS, Gevaert O, Davidzon G, Plevritis SK, West R. NF-κB protein expression associates with (18)F-FDG PET tumor uptake in non-small cell lung cancer: a radiogenomics validation study to understand tumor metabolism. Lung Cancer. 2014;83(2):189–196.
Miao P, Sheng S, Sun X, Liu J, Huang G. Lactate dehydrogenase A in cancer: a promising target for diagnosis and therapy. IUBMB Life. 2013;65(11):904–910.
Zhao J, Huang X, Xu Z, et al. LDHA promotes tumor metastasis by facilitating epithelial–mesenchymal transition in renal cell carcinoma. Mol Med Rep. 2017;16(6):8335–8344.
Lei W, Kang W, Nan Y, et al. The downregulation of miR-200c promotes lactate dehydrogenase A expression and non-small cell lung cancer progression. Oncol Res. Epub 2018 Jan 10.
Dimopoulos MA, Barlogie B, Smith TL, Alexanian R. High serum lactate dehydrogenase level as a marker for drug resistance and short survival in multiple myeloma. Ann Intern Med. 1991;115(12):931–935.
Goldstraw P, Ball D, Jett JR, et al. Non-small-cell lung cancer. Lancet. 2011;378(9804):1727–1740.
Hong LZ, Wei XW, Chen JF, Shi Y. Overexpression of periostin predicts poor prognosis in non-small cell lung cancer. Oncol Lett. 2013;6(6):1595–1603.
Huang B, Zhou H, Lang X, Liu Z. siRNA-induced ABCE1 silencing inhibits proliferation and invasion of breast cancer cells. Mol Med Rep. 2014;10(4):1685–1690.
Zhang T, Zhang DM, Zhao D, et al. The prognostic value of osteopontin expression in non-small cell lung cancer: a meta-analysis. J Mol Histol. 2014;45(5):533–540.
Li T, Zhou J, Deng Z, et al. [Expression of FGF-2 and osteopontin in non-small cell lung cancer]. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2009;34(11):1114–1119. Chinese.
Ostheimer C, Evers C, Bache M, Reese T, Vordermark D. Prognostic implications of the co-detection of the urokinase plasminogen activator system and osteopontin in patients with non-small-cell lung cancer undergoing radiotherapy and correlation with gross tumor volume. Strahlenther Onkol. 2018;194(6):539–551.
Chang YS, Kim HJ, Chang J, Ahn CM, Kim SK, Kim SK. Elevated circulating level of osteopontin is associated with advanced disease state of non-small cell lung cancer. Lung Cancer. 2007;57(3):373–380.
Cui R, Takahashi F, Ohashi R, et al. Abrogation of the interaction between osteopontin and alphavbeta3 integrin reduces tumor growth of human lung cancer cells in mice. Lung Cancer. 2007;57(3):302–310.
Shojaei F, Scott N, Kang X, et al. Osteopontin induces growth of metastatic tumors in a preclinical model of non-small lung cancer. J Exp Clin Cancer Res. 2012;31(1):26.
Lin Q, Guo L, Lin G, et al. Clinical and prognostic significance of OPN and VEGF expression in patients with non-small-cell lung cancer. Cancer Epidemiol. 2015;39(4):539–544.
Jin Y, Tong DY, Tang LY, et al. Expressions of osteopontin (OPN), ανβ3 and Pim-1 associated with poor prognosis in non-small cell lung cancer (NSCLC). Chin J Cancer Res. 2012;24(2):103–108.
Yang Y, Cheng D, Li X, Fang X. [Expression and clinicopathological significance of OPN and CD44v6 in lung cancer]. Zhongguo Fei Ai Za Zhi. 2007;10(2):98–101. Chinese.
Shojaei F, Scott N, Kang X, et al. Osteopontin induces growth of metastatic tumors in a preclinical model of non-small lung cancer. J Exp Clin Cancer Res. 2012;31:26.
Li X, Jiang Z, Li X, Zhang X. SIRT1 overexpression protects non-small cell lung cancer cells against osteopontin-induced epithelial–mesenchymal transition by suppressing NF-κB signaling. Onco Targets Ther. 2018;11:1157–1171.
Kritikou E. Cancer biology: Warburg effect revisited. Nat Rev Mol Cell Biol. 2008;9(4):264.
This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.Download Article [PDF]


text:
Chandrasekhar, M and Khatua, Dipak Kumar and Pattanayak, Ranjit and Kumar, P (2017) Dielectric relaxation and conduction mechanism studies of BNT-BT-BKT ceramics. In: JOURNAL OF PHYSICS AND CHEMISTRY OF SOLIDS, 111 . pp. 160-166.
Jou_Phy_Che_Sol_111_160_2017.pdf - Published Version
Restricted to Registered users only
Download (2MB) | Request a copy
Electrical properties of 0.884Bi(0.5)Na(0.5)TiO(3)-0.036BaTiO(3)-0.08Bi(05)K(0.5)TiO(3) ceramic samples were investigated in 100Hz to 1 MHz frequency range and in 200-450 degrees C temperature range using impedance spectroscopy technique. Real part of impedance versus frequency plots in lower frequency region revealed its PTCR behavior and NTCR behaviors in higher frequency region. Impedance study also suggested the presence of non-Debye type relaxation mechanism. Cole-Cole plots suggested two relaxation regions, in which grain and grain, grain boundary effects were prominent in lower and high temperature regions, respectively. DC conductivity followed Arrhenius law with an activation energy of similar to 0.79 and 1.2 eV, which suggested that the charge carrier were cation vacancies in lower temperature region whereas cation vacancies and oxygen vacancies in higher temperature region.
|Item Type:
|Journal Article
|Additional Information:
|Copy right for this article belongs to the PERGAMON-ELSEVIER SCIENCE LTD, THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, ENGLAND
|Division of Mechanical Sciences > Materials Engineering (formerly Metallurgy)
|Depositing User:
|Id for Latest eprints
|Date Deposited:
|30 Oct 2017 03:41
|Last Modified:
|30 Oct 2017 03:41
Actions (login required)
|View Item