text:
What inspired you to create this month's DST Gold Member kit?
I love decorating my home at Christmas with these woodland Christmas pieces, and I wanted to capture them for a scrap kit.
What is your favorite part or element of the kit?
I especially like the tall Santa and matching snowman.
Tell us about your family: How long have you been designing? What program(s) do you use?
I have the sweetest and most caring husband, two grown children who make me proud, and 5 adorable grandchildren, ages 2 to 7. I’m in Nana heaven when they’re around! I’ve been designing digital scrapkits now for 10 years, and I use almost exclusively PSE5.0. I open the full PhotoShop occasionally, but I’ve learned to trick PSE into doing so many things it didn’t know it could do, and it works more quickly for me that PS.
What part of designing comes easiest to you (themes, colors, papers, elements?)
The colors are easy, but the fun is with the elements. I like to make realistic pieces and also group them in interesting ways.
What part of designing is most challenging to you?
Brand new techniques are always a bit of a struggle for me. You know ... old dog. I do love to learn and improve though.
If you’re running low on design mojo, where do you go to kickstart your creativity?
This rarely ever happens to me. The opposite is usually true where I find myself wanting to design in all directions. I keep a running list of things to design and to take care of behind the scenes, and if I need some inspiration, I just go to my list and start clicking things off. I end up designing and having fun doing it.
What has been the greatest compliment you have received about your designs?
I love hearing from customers, and it’s a real rush to be told that someone looks forward to your next kit. The community is the main reason I continue to design.
Link us to a layout that you have seen using your work that just left you awestruck.
I just love this layout by Robin! The large photo is breathtaking, and I love how she’s included a full photo of this little sweetie as well. She’s such a doll in her boots and hat. Robin made a pretty title and created two clusters from the elements in the kit, Girl Pink.
How much time do you allocate to designing in a typical week?
I design several hours a day and work on marketing another few hours. In any given week, I’d say that I spend at least 50-60 hours on my design business. It’s a good thing that I love what I do!
What is the latest you’ve ever stayed up to finish a design or scrap?
I’ve worked ‘round the clock to finish a collection. Often I just get wrapped up in it and want to see the finished product.
What’s your favorite snack to have handy while you design?
I’m drinking way too many Diet Cokes I know, but I do enjoy having one on my desk at all times.
Slippers or barefoot?
Where is the farthest you have been from home?
I’ve been to Stavanger, Norway, and also to Cairo, Egypt. Egypt is 2,000 miles further from home for me than Norway.


text:
Peanut Butter-Chocolate Pudding Pie
Yield: 16 servings
- Prep Time: 15 minutes
- Cook Time: 20 minutes
- Cooling Time: 4 hours
- Total Time: 4 hours, 35 minutes
For the Crust:
- 1 ¼ cups almond flour
- 5 tablespoons cocoa powder
- ¼ cup Confectioners Swerve
- 4 tablespoons melted butter
- ¼ teaspoon salt
Peanut Butter Layer:
- 8 ounces cream cheese, softened
- ½ cup Confectioners Swerve
- ¾ cup unsweetened creamy peanut butter
- ¾ cup heavy cream, divided
Chocolate Pudding Layer:
- 1 ½ ounces Bake Believe® Semi-Sweet Baking Bar
- 1 cup unsweetened almond milk
- 1 cup heavy cream
- 2 tablespoons unflavored gelatin
- ¾ cup Swerve
- 4 egg yolks
- ½ teaspoon xanthan gum
- ¼ cup cocoa powder
- 3 tablespoons unsalted butter
- ½ teaspoon vanilla
- ¼ teaspoon salt
- ½ ounce Bake Believe® Semi-Sweet Baking Bar, finely chopped
- Preheat the oven to 350°F. Line the bottom of a 9-inch springform pan with parchment paper.
- In a medium bowl, whisk together all of the ingredients for the crust.
- Press the crust ingredients into the bottom of the springform pan, pressing it ½-inch up the sides.
- Bake the crust until aromatic, about 10 minutes.
- Place the crust in the freezer to chill while you make the peanut butter layer.
- Put a medium bowl in the freezer to chill.
- In another medium bowl, beat together the cream cheese, Confectioners Swerve, peanut butter, and ¼ cup of the heavy cream with an electric mixer until well mixed and fluffy.
- Add the remaining ⅓ of the heavy cream to the chilled bowl and beat with an electric mixer until medium peaks form.
- Add the whipped cream to the peanut butter mixture and beat with an electric mixer until well incorporated.
- Spread the peanut butter mixture evenly on top of the prepared crust.
- Chill the pie in the refrigerator while you make the chocolate pudding layer.
- Combine the almond milk and 1 cup heavy cream in a medium saucepan.
- Sprinkle the gelatin on top of the almond milk and cream, then allow to bloom for 10 minutes.
- Place the cream mixture over medium heat, stirring occasionally, until the mixture becomes very hot and begins to steam. Remove from the heat.
- Meanwhile, beat together the ¾ cup Swerve, egg yolks, xanthan gum, and cocoa powder in a medium bowl.
- Continue beating until the mixture becomes very well mixed.
- Add a small amount of the hot liquid to the egg yolk mixture while whisking briskly.
- Continue this until all of the cream mixture has been mixed into the egg yolk mixture.
- Return the cream and yolk mixture to the stove over a medium-low heat.
- Whisk continually until the pudding thickens slightly. Do not allow to boil.
- Remove from the heat and stir in the 1 ½ ounces semi-sweet bar, butter, vanilla, and salt.
- Stir until the butter and semi-sweet bar have melted.
- Place the saucepan in an ice bath and stir until the mixture is lukewarm.
- Pour the chocolate pudding over the peanut butter layer.
- Cover with plastic wrap and press the wrap down onto the surface of the pudding to prevent a skin from forming.
- Chill the pie until the chocolate layer has firmed, about 4 hours.
- Run a small knife around the edge of the pan to loosen the edges.
- Slice the pie into 16 slices and sprinkle with the ½ ounce of semi-sweet baking bar.

domain: food


text:
Are American Football Players Obese?
Dr Haran Sivapalan
February 1, 2019
It's the Super Bowl this weekend and NFL fans are likely to be treated to a veritable feast of sporting prowess. But, did you know that the overwhelming majority of elite American Football players are in fact overweight or obese? They’re also, however, highly-trained, professional and evidently very fit athletes – so, how can they be both these things?
The limits of BMI
As you may have guessed, the discrepancy arises from how we classify ‘overweight’ and ‘obesity’. Both these terms actually (to different degrees) refer to the state of having accumulated an excess amount of fat. Nevertheless, we most commonly use total body mass, or more specifically, Body Mass Index (BMI), as a marker of overweight and obese states. The reason for this is clear - BMI is fairly simple to calculate: you simply divide your body weight (in kilograms) by the square of your height (in metres).
According to the World Health Organisation, a BMI of greater than or equal to 25 kg/m2 is considered overweight, while a BMI greater than or equal to 30 kg/m2 is classified as obese.
But, as the World Health Organisation indeed concede, “BMI should be considered a rough guide because it may not correspond to the same degree of fatness in different individuals.” In other words, by merely measuring bodyweight, BMI does not identify or discriminate between what tissue is truly contributing towards that weight – i.e. is it fat, bone, or muscle? One limitation of BMI then, is that it fails to accurately account for body composition.
The BMI of American football players
Using Body Mass Index as a measure, it is perfectly possible to be classified as overweight or obese under BMI, but, in actuality, have a healthy level of body fat. A large study of Division I collegiate football players highlights this point particularly well.
In the study, all 467 American football players were classified using BMI as overweight or obese. As is to be expected given the different athletic demands of the sport, BMI varied according to which position a subject played. Those in the offensive line had an average BMI of 36.4 kg/m2, placing them in the Class II obese category (BMI between 35.0 and 39.9 kg/m2). By contrast, wide-receivers had an average BMI of 25.7, making them slightly overweight.
Body composition of American football players
As established, however, BMI fails to accurately account for body composition. By contrast, the best currently available method, or ‘gold standard’, for assessing body composition is the DEXA (or DXA) scan.
DEXA stands for ‘Dual Energy X-ray Absorptiometry’. As the name suggests, this scan involves using beams of low-dose X-rays with two distinct energy peaks. As bone, fat and muscle are made from different tissues and have distinguishable consistencies, they absorb the two types of X-ray beams to varying degrees. Using this information, it’s possible to calculate how much of a particular body region is composed of fat, lean muscle and bone.
In the study, collegiate football players also underwent DEXA scans to assess their body composition. More precisely, the researchers measured: total body fat percentage, total lean mass (mass that isn’t fat – i.e. bones, muscle, ligaments and tendons), trunk lean mass, trunk fat mass, legs lean mass, legs fat mass, gluteal (buttocks region) lean mass and visceral adipose tissue mass. Of these measurements, total body fat percentage and visceral adipose tissue are particularly important markers of health status.
Body fat percentage is essentially the proportion of your total weight that is composed of fat. Although there is no official consensus, a healthy body fat percentage is generally thought to be somewhere between 8% and 20% for men, and between 13% and 32% for women. Generally speaking, a higher body fat percentage puts you at a greater risk of diabetes, stroke and heart attack.
When it comes to specific regions of fat that confer these health risks, it is fat tissue found deep in the abdomen and around internal organs that is particularly perilous. This fat tissue is known as visceral adipose tissue.
Returning to American football players, the study found that the vast majority of subjects had healthy body fat percentages (13-20%) and low amounts (<500 grams) of visceral adipose tissue. The only exceptions were offensive and defensive line players, who had average body fat percentages of 31% and 23% respectively. Interestingly, the players also had higher bone mineral density measurements than the general population. This reflects an increased bone mass, which is most likely secondary
So, despite ‘unhealthily’ high BMIs, most collegiate football players are relatively lean, carrying a healthy amount of body fat. As such, they are unlikely to be subject to the same health risks ordinarily implied by having a high BMI.
How do I assess my body composition?
In reality, many of us do not have ready access to advanced DEXA scan technology. Fortunately there are simpler, if slightly less accurate, ways to assess your body fat percentage and body composition.
- Skinfold calipers
This involves measuring the thickness of subcutaneous fat found under the skin using calipers. Multiple skinfold measurements are taken at different regions across the body to get an overview of total body fat percentage.
There are lots of online tutorials available to guide you through the technique. For example, this blog explains how to calculate body fat percentage using skinfold measurements at just 4 body regions.
- Body circumference
The circumference of different parts of your body can also be used to calculate body fat percentage. You’re probably already familiar with waist circumference, which can be a good indicator of the amount of visceral adipose tissue you carry. In addition, the circumference of your neck, hips, wrists and forearms can be assessed.
You can then plug these figures into an online calculator, which uses a formula to give you a body fat percentage. An example online calculator is here.
- Bioelectrical Impedance Analysis (BIA)
You may have spotted a BIA machine in your gym. This technique essentially involves passing a weak electrical current through your body and measuring the ‘impedance’ or opposition to the flow of electricity caused by your body tissues. In general, muscle and lean mass creates less impedance (i.e. it’s easier for electric current to flow through) than fat tissue. Using this information, it’s possible to get an idea of how much body fat you carry.
BIA machines are simple to use and can easily be bought and used at home, but their accuracy is known to vary. Readings may also change according to hydration level, bodyweight (BIA is less accurate in obese people) and intensity of exercise before measurement.

domain: medical


text:
Sunday, June 5, 2022 | 2 a.m.
Why can’t we lobby to allow Texas to secede?
At one time, our democracy was the best and safest place to raise children.
If Texas did not permit buying two assault rifles and nearly 500 rounds bullets without asking any questions as to why this killing gear was being purchased, 21 lives would not have been lost last month in Uvalde.
If an assault rifle was used to down a deer, that deer would be ripped to shreds. Those who had to witness the remains of Uvalde’s children will be traumatized for life.
The bleeding hearts of Texas Republicans will never replace the forever-bleeding hearts of that community.
When the Constitution was drafted, the Second Amendment was concerned about revolutions and single-shot muskets, not an individual with a weapon that could rip children’s heads and bodies apart.

domain: law


November 8, 2018
For immediate release from the UK Communications and Public Relations department.
LEXINGTON, Ky. – Crowned Conference USA Regular-Season Champions last Friday, the No. 3 Kentucky men's soccer team cleaned up in the league's yearly awards on Tuesday.
FB - What It Takes To Win (Louisville)
November 25, 2018
Coach's Corner - UK MBB vs TSU
November 24, 2018
What It Takes To Win (Louisville)
December 2, 2019


text:
James Stevens farmed at Foage, Zennor and Sancreed in the late 19th and early 20th centuries. He kept a Diary in 1877 and from 1892 to 1912, which was transcribed and published in 1977 by the late Peter Pool as A Cornish Farmer’s Diary. I’ve now produced my own edited version of this book, concentrating on entries relating to people and places, and this is available as a free PDF file.
However, if you’d like to read the original 1977 edition, which includes entries relating to farming techniques and practices not reproduced in my edition, it’s possible to buy copies of this book online – full details are…
Stevens, James (1977) A Cornish Farmer’s Diary. Edited by P.A.S. Pool. (ISBN: 0950559601)
… or you could see if your local public library can borrow a copy for you via their interlibrary loan service.
For people researching their family history I’ve put together indexes of over 400 surnames and place names mentioned in the book, plus some businesses. You can view these via the menus above. The Diary could also be useful to anyone with an interest in Cornish local history, historical diaries, old farming techniques, and national and international events of the late Victorian and Edwardian periods. I’ve also produced a volume of ‘supplementary material’, including family trees, which I’m happy to email as a PDF file, also free of charge.
If you’d like to read some entries from the Diary and their background stories, have a look at my posts on the right.
If you’d like to know more (including details of my ‘Diary’ Facebook Group), please contact me via the menu link above.

domain: literature


text: People with clinical manifestations of lymphatic filariasis could be contaminated or not currently. cytokine-producing cells among the three organizations look like because of a subset of cells apart from Compact disc4 T cells. Lymphatic filariasis, which can GM 6001 cell signaling be due to the helminths and it is endemic. Microfilaria matters by GM 6001 cell signaling Nuclepore (Corning-Costar, Cambridge, Mass.) purification of 3 ml of night time bloodstream had been as referred to previously (10). Topics had been categorized into PRKCG three organizations as previously referred to (4). Asymptomatic microfilaremic individuals included subjects with detectable microfilaremia who had no current or previous history of adenolymphangitis, erysipelas, cellulitis, or limb swelling and no physical stigmata of lymphatic GM 6001 cell signaling dysfunction. Antigenemic individuals with clinical filariasis included subjects who had a clinical spectrum of lymphatic pathology ranging from acute filarial fever to chronic lymphedema or elephantiasis, had positive antifilarial immunoglobulin G, and also had current active infection as determined by CAg in serum (TropBio, Townsville, Queensland, Australia) (26). Nonantigenemic individuals with clinical filariasis included subjects with the same clinical manifestations as the antigenemic individuals with clinical GM 6001 cell signaling filariasis but who had undetectable levels of CAg in serum. Seven North American subjects were used as healthy controls. Although not examined in this study, we have previously documented near-universal infection with intestinal helminths in filariasis-endemic communities in Recife and so have assumed our three groups to be matched in this respect. Study patients who were otherwise free of any intercurrent illness got received no diethylcarbamazine therapy within the prior 5 years. Regular diethylcarbamazine treatment was presented with to every single affected person following the scholarly research. Fixation and Excitement of PBMC. PBMC had been isolated by Ficoll-diatrizoate gradient centrifugation from heparinized venous bloodstream. For recognition of intracellular cytokine, PBMC (2 106/ml) had been cultured for 6 h in C-RPMI (RPMI 1640, 10 mM HEPES, 2 mM glutamine, 100 U of penicillin per ml, 100 g of streptomycin per ml, 10% fetal leg serum [FCS]) at 37C and 5% CO2 with or without the current presence of phorbol myristate acetate (PMA; 50 ng/ml; Calbiochem, La Jolla, Calif.) and ionomycin (1 g/ml; Sigma, St. Louis, Mo.). Monensin (1 M; Calbiochem) was contained in all the ethnicities to inhibit cytokine secretion. Following the 6 h of incubation, the cells had been treated with DNase (20 g/ml; Boehringer Mannheim, Indianapolis, Ind.) for 5 min at 37C, cleaned, and set with 4% paraformaldehyde at 37C for 5 min as referred to previously (7). Set cells had been then immediately cleaned with ice-cold phosphate-buffered saline (PBS)C1% bovine serum albumin and kept freezing in PBSC1% bovine serum albuminC10% dimethyl sulfoxide ahead of staining. In a few experiments, Compact disc4 T cells had been negatively chosen from refreshing PBMC as referred to elsewhere (14), utilizing a cocktail of subset-specific antibodies accompanied by two rounds of immunomagnetic adverse selection with goat anti-mouse Fc antibody-conjugated magnetic beads (PerSeptive Diagnostics, Cambridge, Mass., and Dynal, Great Throat, N.Con.). Purity of each sample was evaluated by movement cytometry having a fluorescein isothiocyanate-labeled anti-CD4 antibody (Becton Dickinson, San Jose, Calif.) and found out to become 91.5% 4.7% CD4+. Purified Compact disc4 T cells had been stimulated, set, and frozen beneath the same circumstances as the unfractionated PBMC. Single-cell evaluation of intracellular cytokines. Previously set cells had been washed double with staining buffer (PBSC1% FCSC0.1% sodium azide); 2 105 cells/pipe had been pelleted by centrifugation (250 microfilaremia in men established fact to be considerably higher than in females between your age groups of 15 and 54 in areas encircling the one studied (1). Antigenemic individuals with clinical filariasis had statistically indistinguishable numbers of microfilaria in the blood or levels of circulating antigen compared with asymptomatic microfilaremic individuals. TABLE 1 Characteristics of study?population = 0.02 compared with antigenemic individuals with clinical filariasis and nonantigenemic with clinical filariasis, respectively.? Frequency of cytokine-producing cells in human lymphatic filariasis. To determine whether there were differences in the frequency of IFN–, IL-4-, or IL-5-producing cells in the three patient groups, fresh PBMC were stimulated with PMA-ionomycin or medium alone for 6 h in the presence of monensin and subsequently fixed. Cells were fluorescently stained with either IFN- and IL-4 or IFN- and IL-5 and analyzed by flow cytometry. Figure ?Determine11 illustrates representative two-parameter dot plots for an asymptomatic microfilaremic individual and a nonantigenemic individual with clinical filariasis. The frequencies of IFN–producing and IL-4-producing, but not IL-5-producing, cells are lower in the asymptomatic microfilaremic individual than in the nonantigenemic individual with clinical filariasis. As a group, asymptomatic microfilaremic individuals had significantly lower frequencies of IFN–producing cells compared to nonantigenemic people with scientific filariasis (geometric suggest = 10.71%, range = 4.5 to 18.8 versus.


text:
Go topside to the
Go back to the
or look at the
9/7/91 Princeville, Kauai
A friend was in trouble and needed some inspiration. This is what came through.
TO A FRIEND IN NEED
In this turbulent, challenging time
I thought you might savor a bit of a rhyme.
It's just about flowers that glow in the light,
And stars that shine bright in the dark of the night.
It's only a rhyme about birds that fly free
O'er the peak of a mountain and the deep of a sea.
It's simply a poem about laughter and sharing,
Friendship and loving, healing and caring.


text:
About the Artist
From a humble beginning as a painter dealing with mystical symbolism, Yankel Ginzburg rose to become an internationally recognized artist. As a painter and sculptor of indisputable ability he focuses his talent on monumental canvas paintings, acrylic sculptures and tapestries making him one of the most dynamic and clairvoyant artists in the world today.
Few great artists have successfully mastered more than one medium to express themselves. Yankel Ginzburg is one of them, he glides almost effortlessly between mediums. "I'm a painter who happens to sculpt. When I discovered the powerful prismatic qualities of acrylic forms I became fascinated and excited by the endless possibilities of this medium. Acrylic produces a reflective effect, echoing colors, like no other material. The sculptures I create are powerful, yet warm and vibrant, leaving you with a lasting sense of hope and optimism."
For three decades, Yankel Ginzburg's works have been exhibited the world over. His murals, large tapestries and sculptures are so unique that they are often commissioned for government buildings and association headquarters. Internationally, Yankel Ginzburg's monumental works are well known and often sought after.

domain: artistic


A good friend told me last week to check out the two new singles from Deftones: "Diamond Eyes" and "Rocket Skates," off of their "Diamond Eyes" album which comes out May 4. Boy, do I owe him one! These tracks are off the chain, definitely up there with the band's best. I first heard this band when they got signed to Madonna's Maverick label and their single, "My Own Summer (Shove It)," got a lot of airplay. At the time, they were lumped in with other nu metal and rap metal bands, partially due to Chino's unfortunate "rapping." Most of those bands are gone thankfully, but Deftones keeps moving. Their bass player, Chi Cheng, is unfortunately still in a coma after a car accident in November 2008. Chino Moreno, their lead singer, states they now have two bass players while Cheng recuperates. If you are new to the band, you should check out these two new singles, as well as some of my personal favorites: "Rapture," "Beware" and "Kimdracula" from "Saturday Night Wrist;" "Be Quiet and Drive (Far Away)" and "My Own Summer (Shove It)" from "Around the Fur;" "Bored" from "Adrenaline;" and the entire brilliant "White Pony" record.


text:
Epithelial-mesenchymal transition (EMT) plays an important role in cancer tumorigenesis. However, the underlying mechanisms of EMT in lung adenocarcinoma, and how this process might be inhibited, remain to be explored. This study investigated the role of IL-6 in lung adenocarcinoma cell EMT and explored the potential effects of metformin on this process.
Invasion assay and MTT assay was performed to determine cell invasion and cell proliferation. Western blotting, immunofluorescence, real-time PCR, ELISA, and immunohistochemistry were performed to detect the expression of IL-6, E-cadherin, Vimentin, and p-STAT3.
We discovered that IL-6, via STAT3 phosphorylation, could promote lung adenocarcinoma cell invasion via EMT in vitro. This was supported by the inverse correlation between E-cadherin and IL-6 expression, positive correlation between IL-6 and vimentin mRNA expression and between STAT3 phosphorylation and IL-6 expression in tumor tissues. Importantly, metformin inhibited tumor growth and distant metastases in tumor-bearing nude mice and reversed IL-6-induced EMT both in vitro and in vivo. Furthermore, we found that blockade of STAT3 phosphorylation might be the underlying mechanism of metformin inhibition of IL-6-induced EMT.
Citation: Zhao Z, Cheng X, Wang Y, Han R, Li L, Xiang T, et al. (2014) Metformin Inhibits the IL-6-Induced Epithelial-Mesenchymal Transition and Lung Adenocarcinoma Growth and Metastasis. PLoS ONE 9(4): e95884. https://doi.org/10.1371/journal.pone.0095884
Editor: Srikumar P. Chellappan, H. Lee Moffitt Cancer Center & Research Institute, United States of America
Received: November 14, 2013; Accepted: April 1, 2014; Published: April 30, 2014
Copyright: © 2014 Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by the National Natural Science Foundation of China (No. 81172113, No. 81071912, No. 31270845, No. 81222031), the Chongqing Science Technology Commission (CSTC 2011BB5021), the Chinese “863” Program (No. 2012AA02A201) and National Key Basic Research Program of China (No. 2012CB526603 and No. 2010CB529404). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Non-small cell lung carcinoma (NSCLC) is a leading cause of cancer death worldwide, resulting in an overall 5-year survival rate of less than 15% . Adenocarcinoma accounts for more than half of all NSCLC. Despite advances in treatment, cancer recurrence and formation of distant metastasis are still the main cause of death in patients with lung adenocarcinoma. A better understanding of the mechanisms underlying the formation of distant metastases is required to facilitate the development of effective therapeutic strategies for lung adenocarcinoma patients. Growing evidence reveals that epithelial-mesenchymal transition (EMT) plays a pivotal role in tumorigenesis, drug resistance, relapse and metastasis of various cancers –. Numerous reports have suggested that EMT is a marker of poor prognosis in patients with NSCLC –. Therefore, elucidating the mechanisms underlying EMT and identifying molecular targets and efficient drugs that inhibit this process is a promising approach to attenuate drug resistance, inhibit growth and reduce metastasis, thereby improving the overall survival rates of lung adenocarcinoma patients.
EMT is a complex process, involving dissolution of cell-cell junctions and loss of apical-basolateral polarity, resulting in transition of epithelial cells into migratory mesenchymal cells with invasive properties . Migratory mesenchymal cells after transition are endowed with mesenchymal markers, such as vimentin and N-cadherin, but have lost epithelial markers such as E-cadherin and γ-catenin. Loss of E-cadherin expression is generally accepted as a hallmark of the EMT process . During this process, various transcription factors, such as SNAIL, ZEB1, ZEB2, Twist and E2.2, are key controllers that repress E-cadherin expression. MicroRNAs (miRNAs), such as members of the miR-200 family, are also involved in EMT regulation by targeting the key transcription factors involved in direct repression of E-cadherin, such as ZEB1 and ZEB2 , . The balance between these intrinsic regulators, including both transcription factors and miRNAs, is controlled by extrinsic signals, such as soluble mediators from the tumor microenvironment. Transforming growth factor beta (TGF-β), which is implicated in various tumor metastases, has been identified as the main factor involved in EMT in the tumor microenvironment , .
Interleukin-6 (IL-6) is another key factor in the tumor microenvironment, which is involved in tumorigenesis and progression , . IL-6 activates the IL-6 receptor (IL-6R) to initiate signaling through the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway and also NF-κB . Elevated levels of IL-6 correlate with poor prognosis for a number of types of cancer, such as breast cancer and lung cancer , . Recently, some research groups have reported that IL-6 contributes to tumor metastasis and EMT in breast cancer and ovarian cancer via the JAK/STAT3 signaling pathway , . However, the role of IL-6 during the EMT process in lung adenocarcinoma remains poorly defined.
Metformin, an anti-diabetic drug, is associated with a reduced risk of developing many types of cancer , . Studies have found that treatment of type 2 diabetics with metformin resulted in reduced cancer incidence and improved survival , . Several pharmacological mechanisms may be involved in the anti-tumor function of metformin. Previous studies have found that metformin could inhibit the expression of pro-inflammatory mediators, such as IL-6 and IL-17, which play important roles in tumor development, by reducing activation of NF-κB , . Moreover, metformin could inhibit cell growth and induce apoptosis in triple-negative breast cancers by blocking STAT3 phosphorylation . Thirdly, recent studies have shown that metformin could regulate breast cancer stem cell EMT and significantly down regulate the expression of several EMT markers through decreasing the expression of key drivers of the EMT machinery, such as the transcription factors ZEB1, TWIST1 and Slug . Since IL-6, through JAK/STAT3 signaling, might be involved in EMT in lung adenocarcinoma, and metformin can inhibit EMT and STAT3 phosphorylation in other cancer types, we hypothesized that metformin might be able to inhibit IL-6-induced EMT and growth and metastasis in lung adenocarcinoma.
In this study we provide novel evidence that enhanced IL-6 expression, via STAT3 phosphorylation, is a mechanism that can drive EMT and metastasis in lung adenocarcinoma. Metformin can inhibit IL-6 induced EMT by blocking STAT3 phosphorylation, suggesting a potential clinical use of metformin in the treatment of lung adenocarcinoma.
Materials and Methods
Cell lines and patient samples
Human lung adenocarcinoma cell lines A549 and HCC827 used in this study were purchased from the American Type Culture Collection and were maintained in RPMI 1640 with 10% FBS (Life Technologies, Inc.), 2 mmol/L of l-glutamine (Life Technologies), 100 µg/mL of streptomycin, and 100 units/mL of penicillin in a humidified 5% CO2 atmosphere at 37°C. HCC827-pSB388 cells stably over-expressing recombinant human IL-6 were established by infection of HCC827 cells with lentivirus pSB388, and further selected by puromycin at a concentration of 2 µg/mL, while the control cell line HCC827-pLVT7 was established by infection with lentivirus pLVT7. These cell lines were tested by using short tandem repeat profiling in September, 2012.
Lung adenocarcinoma and corresponding paracancerous tissues from patients treated at the Department of Respiratory Medicine, Daping hospital (Chongqing, China) from Aug 2011 to Sep 2012, were collected by surgical resection. This study was approved by the Institutional Review Board of the Third Military Medical University, China, and prior written and informed consent was obtained from every patient.
Antibodies and drugs
Rabbit monoclonal antibodies against E-cadherin, vimentin, snail and phospho-Stat3 (Tyr705) were purchased from Cell Signaling Technology (Cambridge, MA, USA). Mouse monoclonal antibodies against β-actin and Peroxidase-Conjugated Affinipure goat anti-mouse IgG (H+L) were purchased from ZSBio (Beijing, China). Recombinant human IL-6 was produced by PeproTech (Rocky Hill, NJ). Metformin (Sigma, St. Louis, Missouri, USA) was dissolved in medium and stored at −20°C.
For the matrigel invasion assay, filters (8 µm pore size, Corning Costar Corporation) were precoated with 30 µl Matrigel (BD Biosciences, USA) for 3 h. Cells were starved in serum-free medium overnight, then trypsinized, centrifuged, and resuspended in serum-free RPMI 1640, and placed in the upper wells with 2×104 cells per well supplemented with various concentrations of IL-6 (50, 100 and 500 ng/mL), which was replaced every other day. To determine the effect of metformin on cell invasion, various concentrations (1, 5, and 10 mmol/L) of metformin were added to the culture medium containing 50 ng/ml of IL-6. The lower wells of the transwells contained the same medium as the upper well but with the addition of 2% FBS. After incubation for 18 h, the cells on the upper well and the matrigel were gently removed with a cotton swab, and the cells on the bottom filter were fixed with 4% cold formaldehyde and stained with 0.1% crystal violet. The cells that were attached to the lower surface of the polycarbonate filter were counted under a light microscope (magnification, × 200). Each experiment was repeated three times.
A total of 5000 cells (HCC827-pSB388 and HCC827 cells) were plated in 100 µl medium in 96-well plates. After 48 h incubation, various concentrations of metformin (2.5, 5, 7.5, 10 and 12.5 mmol/L) were added to each well, and cells were further cultured for 48 h. Then 10 µl of 5 mg/ml MTT (Sigma, St. Louis, Missouri, USA) in 100 µl medium was added to each well. After incubation for 4 h, medium was removed and 150 µl of DMSO was added to each well to dissolve the formazan crystals. Then the absorbance at 490 nm was determined using a ThermoFisher Spectrophotometer 1510 (Molecular Devices, Inc.). Cell viability was determined by dividing the absorbance values of treated cells to those of untreated cells.
Immunofluorescence and immunohistochemistry
Immunofluorescence analysis was performed on 8-µm-thick frozen sections, or treated cells. Cells were cultured in 6-well chamber-slides, treated with IL-6 alone or with metformin for the indicated time periods, fixed with ice-cold 4% formaldehyde for 15 minutes at 37°C, and then blocked with rabbit serum for 20 minutes at room temperature before incubation with primary antibodies (E-cadherin 1∶200, vimentin 1∶50) overnight in the dark at 4°C. After three washes, slides were stained with PE-labeled secondary anti-rabbit antibody (1∶500) for 1 h at room temperature and nuclei were counterstained with DAPI. Stained cells were visualized with an Olympus confocal microscope.
Tumor specimens were fixed in 10% formalin, embedded in paraffin, and cut into 6 µm-thick sections. For immunohistochemistry, the sections were deparaffinized, rehydrated, incubated with 3% H2O2 for 10 min, and subjected to heat-induced antigen retrieval by boiling for 10 min in 0.01 M sodium citrate. The specimens were then blocked with normal goat serum for 1 hour and incubated overnight at 4°C with anti-E-cadherin (1∶400), anti-vimentin (1∶100) or anti-phospho-Stat3 (p-Tyr705) antibody (1∶400). The immune complexes were visualized using a kit obtained from the Beyotime Institute of Biotechnology (China) according to the manufacturer's instructions.
Enzyme-linked immunosorbent array (ELISA)
STAT3-pY705 ELISA kit was purchased from Invitrogen Corporation (Invitrogen Corporation, Camarillo, USA). Human IL-6 ELISA Kit was purchased from USCN Life Science Inc (Texas, USA). The levels of STAT3-pY705 and IL-6 in tissue lysates were measured according to the manufacturers' protocols. Absorbance at 450 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). Each measurement was performed in triplicate.
Reverse transcription (RT)-real-time-PCR
Total RNA from cells and tissues was extracted using Trizol Reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer's protocols, and then reverse-transcribed using random hexamers to generate cDNA. Real-time PCR was carried out in 25 µL reactions with 10 pmol primers and the expression levels of E-cadherin, vimentin, snail and IL-6 were detected by real-time PCR using the ABI prism 7300 sequence detection system (Applied Biosystems, New York, USA). Experiments were performed three times in triplicate. The relative mRNA expression levels of E-cadherin, vimentin, snail, and IL-6 were calculated using the comparative Ct (ΔΔCt) method, with GAPDH as a reference gene. The gene-specific primer sequences were shown in Table S1.
Western blot analysis
Cells were lysed with ice-cold M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific Inc, USA) containing PMSF and phosphatase inhibitors for 30 min on ice and then sonicated for 15 seconds to ensure complete cell lysis. Total cellular protein was separated using 8% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Immobilon Transfer Membranes; Millipore). The membranes were blocked with 5% non-fat milk for 1 h, and then incubated with antibodies against p-Stat3 (Tyr705) (1∶2,000), total Stat3 (1∶2,000), E-cadherin (1∶1,000), vimentin (1∶1,000), snail (1∶1,000) and β-actin (1∶2,000) at 4°C overnight. The membrane was then washed three times with 0.1% Tween 20-TBS, and incubated with a horseradish peroxidase-linked secondary antibody (1∶2,000) for 1 h at room temperature. Bands were visualized via enhanced chemiluminescence (ECL) according to the manufacturer's instructions.
In vivo xenograft experiments
A total of 18 six-week-old nude mice (half male and half female) were purchased from the Chinese Academy of Medical Sciences (Beijing, China) and housed and maintained in laminar flow cabinets under specific pathogen-free conditions. These mice were divided into three groups with 6 mice (3 male and 3 female) in each group: group 1 (HCC827) and group 2 (HCC827-pSB388) received pure drinking water, while group 3 (HCC827-pSB388+Met) received drinking water with metformin (250 mg/kg body weight) from 2 days before tumor cell inoculation until the mice were sacrificed. 2×106 HCC827 (group 1) and HCC827-pSB388 (groups 2 and 3) lung cancer cells were injected subcutaneously. Engrafted mice were inspected biweekly for tumor appearance by visual observation and palpation. Tumor volume (mm3) was calculated as length × width2/2. Mouse care and use was performed in strict accordance with the ethical guidelines of the Third Military Medical University. The protocol was approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (Permit Number: SYXK-PLA-2007035). Mice were CO2 -euthanized, and all efforts were made to minimize suffering.
All the data from quantitative assays were expressed as the mean ± standard deviation. Statistical analyses were performed using the independent-samples t-test or one-way ANOVA. The difference was considered statistically significant when p<0.05. All statistical analyses were carried out with SPSS 18.0 software (Chicago, USA).
IL-6 induces EMT and enhances tumor cell invasion in vitro
To determine whether IL-6 is involved in EMT, we first examined the effect of IL-6 on the morphology of lung adenocarcinoma cells. In RPMI 1640 medium, A549 and HCC827 cells exhibited features typical of epithelial cells, displaying a distinctive cobblestone appearance and forming clusters. However, after stimulation with 100 ng/mL IL-6 for 1 week, both A549 and HCC827 cells became scattered and acquired fibroblast-like shapes, which are characteristics of mesenchymal-like morphology (Fig. 1A). These changes in morphology suggest that A549 and HCC827 cells might undergo EMT in response to IL-6 stimulation. It has been established that mesenchymal cells are endowed with enhanced invasion and metastatic capabilities. Therefore, we determined the effect of IL-6 on the cells' invasion abilities. The results showed that IL-6 treatment could significantly enhance invasive capacity in a dose-dependent manner for both A549 and HCC827 cell lines (Fig. 1B and Fig. S1). Importantly, Western blotting (Fig. 1C), quantitative PCR (Fig. 1D) and immunofluorescence (Fig. 1E) showed that treatment of both A549 and HCC827 cells with 50 ng/mL IL-6 for 1 week reduced the expression of an epithelial cell marker (E-cadherin), and increased the expression of mesenchymal cell markers (vimentin and snail). Collectively, the alterations in cell morphology, increased invasive capacity, acquisition of mesenchymal cell markers and loss of epithelial cell markers indicate that lung adenocarcinoma cells undergo EMT upon IL-6 stimulation.
(A) A549 and HCC827 cell mesenchymal phenotype induced by IL-6 (200×). (B) A549 and HCC827 cell invasion in the presence of various concentrations of IL-6. (C) E-cadherin, vimentin and snail protein levels were examined by Western blotting. Equal protein loading was verified by β-actin. (D) mRNA expression levels of E-cadherin, vimentin and snail were analyzed by quantitative PCR and normalized by GAPDH. (E) E-cadherin and vimentin protein levels were detected by immunofluorescence (800×). Error bars represent the standard deviation. *, p<0.05, **, p<0.005.
A negative correlation between IL-6 production and E-cadherin expression, and a positive correlation between IL-6 production and vimentin expression are detectible in lung adenocarcinoma tissues
As we demonstrated that stimulation with IL-6 in vitro could induce EMT in lung adenocarcinoma cell lines, we speculate that there may be a correlation between IL-6 production and mesenchymal or epithelial cell markers in lung adenocarcinoma tissues. Accordingly, we examined the expression of IL-6, E-cadherin and vimentin mRNA in tissue samples from 18 cases of lung adenocarcinoma, and 6 corresponding paracancerous tissues, by quantitative PCR, and analyzed the correlation between IL-6 and E-cadherin or vimentin expression. As expected, we found that IL-6 mRNA expression in lung adenocarcinoma tissues was much higher than in paracancerous tissues (p<0.005, Fig. 2A), similar to results reported previously , . Importantly, there was a negative correlation between IL-6 and E-cadherin expression (Fig. 2B), and a positive correlation between IL-6 and vimentin expression (Fig. 2C). We also used immunofluorescence assays to determine the protein levels of E-cadherin and vimentin in lung adenocarcinoma tissue samples with low IL-6 or high IL-6 expression. The results showed weak vimentin expression but strong E-cadherin expression in low IL-6-expressing cancer tissues, whereas strong vimentin expression but weak E-cadherin expression was observed in high IL-6-expressing cancer tissues (Fig. 2D). The correlation between IL-6 and EMT markers in clinical samples, together with the in vitro IL-6 stimulation results, strongly suggests that IL-6 is involved in EMT in lung adenocarcinoma.
(A) IL-6 gene expression levels in tumor (n = 18) and corresponding paracancerous (n = 6) tissues was analyzed by real-time PCR, with GAPDH as a reference gene. p<0.005. (B) IL-6 and E-cadherin gene expression in tumor tissues was analyzed by real-time PCR and the correlation between these two genes was analyzed. p<0.05. (C) IL-6 and vimentin gene expression in tumor tissues was analyzed by real-time PCR and the correlation between these two genes was analyzed (p<0.0001). (D) Representative immunofluorescence staining for E-cadherin and vimentin in lung adenocarcinoma tissues with low IL-6 or high IL-6 expression (800×).
IL-6 activates STAT3 phosphorylation in vitro and a positive correlation between IL-6 production and STAT3 phosphorylation is detected in lung adenocarcinoma tissues
The involvement of the JAK-STATs signaling pathway in IL-6/IL-6R signaling and involvement of STAT3 phosphorylation in EMT of cancer cells has been proved by lots of results , , . Therefore, we speculate that the IL-6-induced EMT of lung adenocarcinoma cells may be due to activation of STAT3. To investigate this point, we first examined the effect of IL-6 on STAT3 tyrosine phosphorylation in A549 and HCC827 lung adenocarcinoma cells. As expected, we found that 50 ng/mL IL-6 activated STAT3 and induced STAT3 tyrosine phosphorylation on Tyr 705 in A549 and HCC827 cells. STAT3 was rapidly phosphorylated upon IL-6 treatment, reaching the highest level at the 30 minute time point, and then declined to the basal level at 120 minutes (Fig. 3A). We also detected the STAT3 phosphorylation in A549 and HCC827 cells treated with 50 ng/mL of IL-6 for 1 week. The results showed that high STAT3 phosphorylation level was detected after IL-16 treatment for 1 week (left panel, Fig. 3B). Moreover, we detected STAT3 phosphorylation in HCC827-pSB388 cells, which stably over expressed IL-6. Consistent with IL-6 stimulation, HCC827-pSB388 cells, but not HCC827 or HCC827-pLVT7 cells, displayed STAT3 phosphorylation (right panel, Fig. 3B). Importantly, the level of IL-6 was positively associated with the level of STAT3 phosphorylation in lung adenocarcinoma tissues when the values for each individual patient were plotted (p<0.005, Fig. 3C). Moreover, we found significantly higher STAT3 phosphorylation in high IL-6-producing tumor tissues than that in low IL-6-producing tissues (Fig. 3D–E). Again, these data provide strong support for our hypothesis that induction of EMT by IL-6 is through activation of STAT3.
(A) STAT3 phosphorylation status in A549 and HCC827 cells after stimulation with IL-6 in a short time. (B) STAT3 phosphorylation status was determined by western blotting. A549 and HCC827 cells were stimulated with IL-6 in a long time (left). HCC827-pSB388 cells were stimulated with IL-6. HCC827 and HCC827-pLVT7 cells were used as a control (right). (C) Correlation between IL-6 production and STAT3 phosphorylation in human lung adenocarcinoma tissues (n = 18, p<0.0001). (D) Representative immunohistochemical staining for STAT3 phosphorylation in lung adenocarcinoma tissues with low IL-6 or high IL-6 expression (400 ×). (E) Comparison of the score of p-STAT3 with different IL-6 expression level.
Metformin inhibits IL-6-induced EMT in lung adenocarcinoma cells
Since TGF-β is involved in EMT, TGF-β-neutralizing antibodies and TGF-β receptor inhibitors have been employed in attempts to inhibit EMT, thereby reducing tumor metastasis and enhancing chemotherapy sensitivity , . Our discovery of the involvement of IL-6 in the induction of EMT provides an impetus to find a drug to block IL-6-induced EMT. Previous studies have shown that metformin could inhibit STAT3 phosphorylation, which is involved in the IL-6/IL-6R signaling pathway and, as we have shown, IL-6-induced EMT. Therefore, we hypothesize that metformin might be a potential drug to inhibit IL-6-induced EMT. To investigate this, we first determined the effect of metformin on IL-6-induced EMT in vitro. Consistent with our hypothesis, our data revealed that 5 mmol/L (p<0.05) or 10 mmol/L (p<0.005) metformin could significantly reduce lung cancer cell invasion, which was induced by IL-6 (Fig. 4A and Fig. S2) in both A549 and HCC827 cell lines. The extent of metformin inhibition of invasion at 10 mmol/L was similar to that seen with 2 µmol/L cucurbitacin Q, a specific STAT3 inhibitor (Fig. 4A). Additionally, quantitative PCR (Fig. 4C), Western blotting (Fig. 4B) and immunofluorescence (Fig. 4D) showed that metformin could reverse the IL-6 treatment-induced changes, significantly repressing vimentin and snail expression, and restoring E-cadherin expression. Collectively, these in vitro results showed that metformin inhibits IL-6-induced EMT in lung adenocarcinoma cells.
(A) Invasion of IL-6 treated lung cancer cells in the presence of various concentrations of metformin. The selective STAT3 inhibitor cucurbitacin Q (Cuc, 2 µmol/L) served as a positive control for inhibition of lung cancer cell invasion. (B) Protein expression levels of E-cadherin, vimentin and snail in cells treated with IL-6 alone or in combination with metformin were analyzed by western blotting. β-actin was used as a loading control. (C) mRNA expression levels of E-cadherin, vimentin and snail in cells treated with IL-6 alone or in combination with metformin were examined by real time PCR and normalized by GAPDH. (D) The expression of E-cadherin and vimentin was detected by immunofluorescence in A549 and HCC827 cells treated with IL-6 alone or in combination with metformin (800×). Error bars represent the standard deviation (*, p<0.05; **, p<0.005).
Metformin suppresses IL-6-enhanced tumor growth and metastasis in nude mice
To examine the potential role of metformin in tumor growth and metastasis of lung adenocarcinoma, we first established stable IL-6-expressing HCC827 cells (HCC827-pSB388) by lentivirus infection. This was necessary since IL-6 in the tumor tissues is mainly produced by infiltrating inflammatory cells and, being of mouse origin, this IL-6 will have no effect on human tumor cells. We then examined the expression of E-cadherin and vimentin protein in HCC827-pLVT7 and HCC827-pSB388 cells by Western blotting, and the cells' invasive capacity. The results showed that the over-expression of IL-6 in HCC827-pSB388 cells could repress E-cadherin expression and elevate vimentin expression, and also enhanced the cells' invasive capacity in vitro (Fig. S3). These results were consistent with the results from the IL-6 stimulation experiments.
We next investigated the effect of IL-6 expression on tumor growth and metastasis in vivo using xenograft experiments. We found that IL-6 transfection significantly promoted the growth of HCC827 tumors (Fig. 5A–B), consistent with the previous findings of a pro-tumor activity of IL-6 in lung adenocarcinoma . However, this enhancement of tumor growth by IL-6 transfection was partially reversed by metformin treatment (Fig. 5A–B), indicating an inhibitory effect of metformin on IL-6-induced tumor growth. Importantly, we found significantly fewer metastatic nodes in lung and liver tissues from metformin-treated HCC827-pSB388-bearing mice than in PBS-treated HCC827-pSB388-bearing mice, whereas there was no metastatic node from HCC827-bearing mice (p<0.05) (Fig. 5C–D). In addition, using MTT, we found that metformin significantly decreased the proliferation of both HCC827 cells and HCC827-pSB388 cells (Fig. S4).
(A) Xenograft at sacrifice. Xenografts from HCC827-pSB388 group were much larger than HCC827 group. Xenografts from HCC827-pSB388+Met group, which treated with metformin, were much smaller than that from HCC827-pSB388 group. (B) Tumor volumes were determined at the time of sacrifice. (C) Metastatic tumor nodules in the lung were examined by H & E staining of serial sections. Tumor nodules are marked with red arrows (100× and 400×). (D) The numbers of cancerous metastatic nodules in these lung sections were counted and the average number per field of view is presented. (E) E-cadherin and vimentin expression in tumor tissues from HCC827, HCC827pSB388 and HCC827pSB388+Met groups was analyzed by immunohistochemistry (400×). Error bars represent the standard deviation (*, p<0.05).
Given the in vitro evidence that metformin could inhibit EMT of lung adenocarcinoma cells, we suppose that the reduction in metastasis formation seen with metformin was due to inhibition of HCC827-pSB388 cell EMT. Consequently, we examined the expression of EMT markers in HCC827, HCC827-pSB388 and metformin-treated HCC827-pSB388 tumor tissues with immunohistochemistry. As expected, we found reduced E-cadherin expression and enhanced vimentin expression in HCC827-pSB388 tumors compared with HCC827 tumors, further supporting the in vitro findings that IL-6 could induce EMT of lung adenocarcinoma cells. Interestingly, metformin could partially decrease EMT caused by IL-6 over expression (Fig. 5E). These results suggest that metformin inhibits tumor growth and metastasis of lung adenocarcinoma cells in xenografted mice via inhibition of EMT.
Metformin inhibits lung adenocarcinoma EMT through inhibition of IL-6-induced STAT3 tyrosine phosphorylation
To elucidate the underlying mechanisms of metformin inhibition of IL-6-induced EMT, we first determined the effect of metformin on IL-6-induced STAT3 tyrosine phosphorylation. We added 1–10 mmol/L metformin to A549 and HCC827 cells cultured in medium containing 50 ng/mL of IL-6, and to HCC827-pSB388 cells for 1 week. The results showed that metformin treatment could significantly reduce IL-6-induced STAT3 phosphorylation in A549, HCC827 and HCC827-pSB388 lung adenocarcinoma cells in a dose-dependent manner (Fig. 6A-B). Immunofluorescence assay on HCC827-pSB388 cells further showed that metformin inhibits STAT-3 phosphorylation and the effect is similar to a specific STAT3 inhibitor, cucurbitacin Q (Fig. 6C). These results suggest that metformin inhibits EMT of lung adenocarcinoma cells through inhibition of IL-6-induced STAT3 tyrosine phosphorylation in vitro. Next we examined STAT3 phosphorylation in tumors from HCC827 and HCC827-pSB388 cell grafted mice by immunohistochemistry. We found more STAT3 phosphorylation in tumors from HCC827-pSB388 cell grafted mice than in tumors from HCC827 cells grafted mice (Fig. 6D-E, p<0.001). This result was consistent with the findings from clinical samples, which showed a positive correlation between IL-6 production and STAT3 phosphorylation (Fig. 3C–E). However, the phosphorylation of STAT3 was significantly reduced in tumors from HCC827-pSB388 cell grafted mice that were treated with metformin (Fig. 6D–E). These results suggest that inhibition of STAT3 phosphorylation might be the underlying mechanism of metformin inhibition of IL-6-induced EMT.
(A) Lung cancer cells were treated with IL-6 and the indicated concentrations of metformin, STAT3 phosphorylation status was determined by western blotting. Metformin suppressed STAT3 phosphorylation induced by IL-6 in lung adenocarcinoma cells in a dose-dependent manner. (B) CC827-pSB388 cells were treated with the indicated concentrations of metformin and STAT3 phosphorylation was determined by western blotting. (C) STAT3 phosphorylation in untreated HCC827-pSB388 cells, HCC827-pSB388 cells treated with metformin and HCC827-pSB388 cells treated with cucurbitacin Q were examined by immunofluorescence (800×). (D) STAT3 phosphorylation in tumors from HCC827, HCC827-pSB388 and HCC827-pSB388+Met groups was examined by immunohistochemistry (400×). (E) The average number of p-STAT3 positive cells in each field of view was analyzed. Error bars represent the standard deviation (**, p<0.005).
Further, to determine the effect of metformin on AMPK activation in HCC827 cells and HCC827-pSB388 cells, Western blot analysis was performed. As shown in Fig. S5, Western Blot results showed that expression of phosphorylated AMPK in HCC827-pSB388 cells was lower when compared to that of HCC827 cells. Metformin treatment significantly increased activation of AMPK in HCC827-pSB388 cells (Fig. S5).
EMT is an important biological process that plays a critical role in tumorigenesis. However, the mechanisms underlying EMT in lung adenocarcinoma and how this process is inhibited remain to be explored. In this study, we showed that IL-6 was involved in EMT and metastasis, via induction of STAT3 phosphorylation in lung adenocarcinoma cells, by in vitro IL-6 stimulation experiments, clinical correlation analysis and xenograft experiments. In addition, we found that metformin could inhibit IL-6 enhancement of tumor growth, as well as reduce tumor metastasis, through inhibition of STAT3-mediated EMT. Collectively, these results not only suggest that the IL-6/IL-6R/STAT3 may be used as molecular targets for the inhibition of IL-6-induced EMT, but also provide experimental evidence for the potential use of metformin in the treatment of lung adenocarcinoma.
It is well known that inflammatory mediators and cells are involved in the migration, invasion and metastasis of malignant cells . However, most studies into the mechanisms of inflammation-driven metastasis have focused on the induction of chemokine receptors, which can direct the movement of cancer cells, by inflammatory cytokines such as TNF-α, IL-1β and IL-6 , . Growing evidence has suggested that inflammatory mediators, produced by tumor-infiltrating leukocytes and cancer-related fibroblasts, also promote tumor metastasis via inducing EMT –. Numerous reports indicate that growth factors, including TGF-β, epidermal growth factor (EGF), vascular endothelial growth factor, platelet-derived growth factor and hepatocyte growth factor can induce cancer cell EMT in a variety of cancers including lung adenocarcinoma . Recently, another inflammatory cytokine IL-6 has been reported as a potent inducer of EMT in breast cancer cells with an epithelial phenotype . Previous studies have found IL-6 in the serum of approximately 40% of patients with NSCLC , . Koh and colleagues found that the expression of IL-6 in tumor tissues correlated with the concentration of serum IL-6, tumor progression, and the overall survival in NSCLC . Taken together, these findings suggest a role for IL-6 in the progression of lung adenocarcinoma. In this study, we demonstrated that IL-6 could drive EMT and promote metastasis in lung adenocarcinoma, similar to the function of IL-6 in breast cancer and head and neck tumor metastasis.
Mesenchymal-like cancer cells in lung adenocarcinoma are endowed with enhanced invasive ability and resistance to chemotherapy and tyrosine kinase inhibitors (TKI) . Therefore, pharmacologists are focusing on the identification of EMT inhibitors. Since the role of TGF-β in EMT was first recognized, TGF-β-neutralizing antibodies and TGF-β receptor inhibitors have been studied to inhibit EMT , . Recently, metformin, which is widely used as first-line drug for type 2 diabetes, is found to repress TGF-β-induced EMT in breast cancer . However, whether metformin could also inhibit IL-6-induced EMT remains to be explored. In this study, we found that metformin could not only retard IL-6 enhancement of tumor growth, but also reduce tumor metastasis in lung adenocarcinoma cell-bearing nude mice. Importantly, we found that metformin partially inhibited IL-6-induced EMT and induced the reacquisition of an epithelial phenotype as characterized by the gain of epithelial marker expression and decreased expression of mesenchymal markers. To our knowledge, this was the first study that showed metformin could inhibit IL-6-mediated EMT and tumor metastasis. Interestingly, metformin inhibited tumor cell growth in vitro and tumor growth in vivo. Thus, the inhibitory effect of metformin on tumor metastasis might be due to the reduced number of tumor cells induced by metformin. We do not rule out this possibility. However, the relationship between the effect of metformin on tumor metastasis and the effect of metformin on tumor growth still needs further investigation.
IL-6/IL-6R performs biological functions mainly through JAK-STAT3. It has been reported that the activation of STAT3 in tumor cells promotes tumor growth by increasing the capacity of tumors to evade the immune system , . Additionally, STAT3 is involved in EMT in ovarian cancer, and STAT3 phosphorylation is significantly correlated with TNM (tumor, lymph node, and metastasis stages) . In addition, previous results have revealed metformin could block STAT3 phosphorylation in triple-negative breast cancers . In this study, we found that metformin treatment inhibited STAT3 phosphorylation and induced AMPK phosphorylation in HCC827 cells and HCC827-pSB388 cells. It is reported that activated AMPK can suppress STAT3 signaling and STAT3 phosphorylation , . Although metformin may directly target STAT3 , it is still possible that the effect of metformin on STAT3 phosphorylation may be indirectly mediated through AMPK activation. However, the direct effect and mechanism of AMPK activation induced by metformin on STAT3 in lung cancer cells still needs further investigation. Based on the knowledge mentioned above and our new findings that metformin repressed the IL-6-activated STAT3 phosphorylation, we suggest that metformin inhibits IL-6-induced EMT and metastasis of lung adenocarcinoma cells via blockade of STAT3 phosphorylation. However, we cannot exclude the possibility of other potential mechanisms contributing to the inhibition of IL-6-induced EMT by metformin.
In conclusion, we demonstrated that enhanced IL-6 production, via STAT3 phosphorylation, was one of the underlying mechanisms of EMT and metastasis in lung adenocarcinoma. We found that metformin could inhibit IL-6 induced EMT possibly by blocking STAT3 phosphorylation, suggesting a potential clinical use of metformin in treatment of lung adenocarcinoma.
IL-6 promoted A549 and HCC827 cells invasion. A549 and HCC827 cells invasion was promoted by various concentrations IL-6 (200×).
Metformin inhibited A549 and HCC827 cells invasion. Metformin inhibited A549 and HCC827 cells invasion, which was induced by 50 ng/ml IL-6 at the various concentrations. As a positive control, Cuc (2 µmol/L), a p-STAT3 special inhibitor, inhibited A549 and HCC827 cells invasion (200×).
Overexpressed IL-6 promoted HCC827 cell invasion and EMT. (A) Over-expression of IL-6 was verified by western blotting. No IL-6 expression was detected in the control cell line HCC827 and the negative control cell HCC827-pLVT7. There was a strong IL-6 expression in the IL-6 overexpression cell HCC827-pSB388. GAPDH was used as a loading control. (B) Over-expression of IL-6 promoted invasion of HCC827 cells (400×). (C) The numbers of the invasion cells were statistically analyzed. Error bars represent the standard deviation (*, p<0.05). (D) The E-cadherin and vimentin expression in cell HCC827-pSB388 was analyzed by western blotting. Compared with HCC827 and HCC827-pLVT7, overexpression of IL-6 repressed E-cadherin expression and elevated vimentin expression. β-actin was used as a loading control.
Metformin decreased proliferation of both HCC827 cells and HCC827-pSB388 cells. Cells were untreated or treated with different doses of metformin as indicated for 48 hours. Cell viability was assayed using MTT method. Data was expressed as Mean ± SEM. *, p<0.05, #, p<0.01.
Metformin activation of AMPK in HCC827-pSB388 cells. Expression of phosphoralylated AMPK and total AMPK were detected using Western blot analysis in HCC827 cells and HCC827-pSB388 cells, untreated or treated with metformin. β-actin was used as a loading control.
We appreciate Dr. Symonds A.L.J (Institute of Cellular and Molecular Science, University of London, UK. A.L.J. (Symonds@qmul.ac.uk) for the revision and correction of this manuscript.
Conceived and designed the experiments: BZ YH. Performed the experiments: ZZ. Analyzed the data: XC. Contributed reagents/materials/analysis tools: YW RH LL TX LH HL. Wrote the paper: ZZ XC BZ.
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- 27. Martin-Castillo B, Vazquez-Martin A, Oliveras-Ferraros C, Menendez JA (2010) Metformin and cancer: doses, mechanisms and the dandelion and hormetic phenomena. Cell Cycle 9: 1057–1064.
- 28. Nath N, Khan M, Paintlia MK, Singh I, Hoda MN, et al. (2009) Metformin attenuated the autoimmune disease of the central nervous system in animal models of multiple sclerosis. J Immunol 182: 8005–8014.
- 29. Isoda K, Young JL, Zirlik A, MacFarlane LA, Tsuboi N, et al. (2006) Metformin inhibits proinflammatory responses and nuclear factor-kappaB in human vascular wall cells. Arterioscler Thromb Vasc Biol 26: 611–617.
- 30. Deng XS, Wang S, Deng A, Liu B, Edgerton SM, et al. (2012) Metformin targets Stat3 to inhibit cell growth and induce apoptosis in triple-negative breast cancers. Cell Cycle 11: 367–376.
- 31. Vazquez-Martin A, Oliveras-Ferraros C, Cufi S, Del BS, Martin-Castillo B, et al. (2010) Metformin regulates breast cancer stem cell ontogeny by transcriptional regulation of the epithelial-mesenchymal transition (EMT) status. Cell Cycle 9: 3807–3814.
- 32. Xiong H, Hong J, Du W, Lin YW, Ren LL, et al. (2012) Roles of STAT3 and ZEB1 proteins in E-cadherin down-regulation and human colorectal cancer epithelial-mesenchymal transition. J Biol Chem 287: 5819–5832.
- 33. Huang C, Yang G, Jiang T, Zhu G, Li H, et al. (2011) The effects and mechanisms of blockage of STAT3 signaling pathway on IL-6 inducing EMT in human pancreatic cancer cells in vitro. Neoplasma 58: 396–405.
- 34. Hawinkels LJ, Ten DP (2011) Exploring anti-TGF-beta therapies in cancer and fibrosis. Growth Factors 29: 140–152.
- 35. Bhola NE, Balko JM, Dugger TC, Kuba MG, Sanchez V, et al. (2013) TGF-beta inhibition enhances chemotherapy action against triple-negative breast cancer. J Clin Invest 123: 1348–1358.
- 36. Yamaji H, Iizasa T, Koh E, Suzuki M, Otsuji M, et al. (2004) Correlation between interleukin 6 production and tumor proliferation in non-small cell lung cancer. Cancer Immunol Immunother 53: 786–792.
- 37. Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G (2006) Inflammation and cancer: how hot is the link?. Biochem Pharmacol 72: 1605–1621.
- 38. Balkwill F, Charles KA, Mantovani A (2005) Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 7: 211–217.
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- 40. Soria G, Ofri-Shahak M, Haas I, Yaal-Hahoshen N, Leider-Trejo L, et al. (2011) Inflammatory mediators in breast cancer: coordinated expression of TNFalpha & IL-1beta with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition. BMC Cancer 11: 130.
- 41. Dohadwala M, Wang G, Heinrich E, Luo J, Lau O, et al. (2010) The role of ZEB1 in the inflammation-induced promotion of EMT in HNSCC. Otolaryngol Head Neck Surg 142: 753–759.
- 42. Mimeault M, Batra SK (2007) Interplay of distinct growth factors during epithelial mesenchymal transition of cancer progenitor cells and molecular targeting as novel cancer therapies. Ann Oncol 18: 1605–1619.
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- 46. Frederick BA, Helfrich BA, Coldren CD, Zheng D, Chan D, et al. (2007) Epithelial to mesenchymal transition predicts gefitinib resistance in cell lines of head and neck squamous cell carcinoma and non-small cell lung carcinoma. Mol Cancer Ther 6: 1683–1691.
- 47. Cufi S, Vazquez-Martin A, Oliveras-Ferraros C, Martin-Castillo B, Joven J, et al. (2010) Metformin against TGFbeta-induced epithelial-to-mesenchymal transition (EMT): from cancer stem cells to aging-associated fibrosis. Cell Cycle 9: 4461–4468.
- 48. See AP, Han JE, Phallen J, Binder Z, Gallia G, et al. (2012) The role of STAT3 activation in modulating the immune microenvironment of GBM. J Neurooncol 110: 359–368.
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- 51. Li H, Lee J, He C, Zou MH, Xie Z (2014) Suppression of the mTORC1/STAT3/Notch1 pathway by activated AMPK prevents hepatic insulin resistance induced by excess amino acids. Am J Physiol Endocrinol Metab 306: E197–209.
- 52. Nerstedt A, Johansson A, Andersson CX, Cansby E, Smith U, et al. (2010) AMP-activated protein kinase inhibits IL-6-stimulated inflammatory response in human liver cells by suppressing phosphorylation of signal transducer and activator of transcription 3 (STAT3). Diabetologia 53: 2406–2416.


text:
Happy Friday everyone! and well done on making it to the end of the week....
I am so pleased to be able to bring you this amazing post full of so much pretty to the blog. Debbie is a great friend of Kelly H’s and she is such an amazing designer. I have had the pleasure last year of photographing some of Debbie’s pieces at the weddings I have attended…. Read more »
Today on Bridal Style, I would like to introduce you to Nymphi, a new bridal collection from Greece which is soon to launch in the UK. ‘The collection will include, veils, floral head pieces, sashes, cuffs, collars and shoulder pieces, Nymphi’s bridal collection has much to offer. With a range of look defining, statement pieces and a variety… Read more »


text:
brought us the German moment, in which Jewish philosophy was framed and
deeply shaped by the thought of Kant and Hegel (we should add that the “
German moment” actually included important influences from France, including
Author: Samuel Lebens
Publisher: Oxford University Press, USA
Since the classical period, Jewish scholars have drawn on developments in philosophy to enrich our understanding of Judaism. This methodology reached its pinnacle in the medieval period with figures like Maimonides and continued into the modern period with the likes of Rosenzweig. The explosion of Anglo-American/analytic philosophy in the twentieth century means that there is now a host of material, largely unexplored by Jewish philosophy, with which to explore, analyze, and develop the Jewish tradition. Jewish Philosophy in an Analytic Age features contributions from leading scholars in the field which investigate Jewish texts, traditions, and/or thinkers, in order to showcase what Jewish philosophy can be in an analytic age. United by the new and engaging style of philosophy, the collection explores rabbinic and Talmudic philosophy; Maimonidean philosophy; philosophical theology; and ethics and value theory.
Important works include Isaac Husik, A History of Mediaeval Jewish Philosophy (
Philadelphia, PA: Jewish Publication Society of America, 1916); Julius Guttmann,
Philosophies of Judaism: A History of Jewish Philosophy from Biblical Times to ...
Author: Aaron W. Hughes
Publisher: Indiana University Press
Too often the study of philosophical texts is carried out in ways that do not pay significant attention to how the ideas contained within them are presented, articulated, and developed. This was not always the case. The contributors to this collected work consider Jewish philosophy in the medieval period, when new genres and forms of written expression were flourishing in the wake of renewed interest in ancient philosophy. Many medieval Jewish philosophers were highly accomplished poets, for example, and made conscious efforts to write in a poetic style. This volume turns attention to the connections that medieval Jewish thinkers made between the literary, the exegetical, the philosophical, and the mystical to shed light on the creativity and diversity of medieval thought. As they broaden the scope of what counts as medieval Jewish philosophy, the essays collected here consider questions about how an argument is formed, how text is put into the service of philosophy, and the social and intellectual environment in which philosophical texts were produced.
The idea of placing histories of Islamic and Jewish philosophy in such close
proximity to a history of Western philosophy is in our view timely and important.
Jewish and Islamic philosophy are often viewed as mere footnotes in general
Author: Daniel Frank
Jewish philosophy is often presented as an addendum to Jewish religion rather than as a rich and varied tradition in its own right, but the History of Jewish Philosophy explores the entire scope and variety of Jewish philosophy from philosophical interpretations of the Bible right up to contemporary Jewish feminist and postmodernist thought. The links between Jewish philosophy and its wider cultural context are stressed, building up a comprehensive and historically sensitive view of Jewish philosophy and its place in the development of philosophy as a whole. Includes: · Detailed discussions of the most important Jewish philosophers and philosophical movements · Descriptions of the social and cultural contexts in which Jewish philosophical thought developed throughout the centuries · Contributions by 35 leading scholars in the field, from Britain, Canada, Israel and the US · Detailed and extensive bibliographies
Identifies and examines the central insights of Franz Rosenzweig and Emmanuel Levinas concerning the religious dimensions of the relationships between persons and extends these insights in order to explore the relevance of religious ...
Author: Michael Oppenheim
Publisher: SUNY Press
Identifies and examines the central insights of Franz Rosenzweig and Emmanuel Levinas concerning the religious dimensions of the relationships between persons and extends these insights in order to explore the relevance of religious language to speak of post-Holocaust Jewish life, the critique of the tradition by feminist Jewish philosophers and theologians, and the challenges of religious pluralism.
enton] Was Hermann Cohen a Jewish thinker or a German philosopher? Did he
belong chiefly to the tradition of Jewish philosophy or instead to the classical
tradition of critical idealism, of which Kant is the paradigmatic figure and
Author: Michael L. Morgan
Publisher: Cambridge University Press
Modern Jewish philosophy emerged in the seventeenth century, with the impact of the new science and modern philosophy on thinkers who were reflecting upon the nature of Judaism and Jewish life. This collection of essays examines the work of several of the most important of these figures, from the seventeenth to the late-twentieth centuries, and addresses themes central to the tradition of modern Jewish philosophy: language and revelation, autonomy and authority, the problem of evil, messianism, the influence of Kant, and feminism. Included are essays on Spinoza, Mendelssohn, Cohen, Buber, Rosenzweig, Fackenheim, Soloveitchik, Strauss, and Levinas. Other thinkers discussed include Maimon, Benjamin, Derrida, Scholem, and Arendt. The sixteen original essays are written by a world-renowned group of scholars especially for this volume and give a broad and rich picture of the tradition of modern Jewish philosophy over a period of four centuries.
See the comments in Seeskin, Autonomy in Jewish Philosophy, 178. 20. Cohen,
Religion of Reason out of the Sources of Judaism, 147. 21. Bergman, Dialogical
Philosophy from Kierkegaard to Buber, 153-154. 22. E.g., Religion of Reason out
Author: Aaron W. Hughes
Jewish philosophy as a dynamic literary, cultural, and social practice
A Chomprehensive anthology of classic writings on Jewish philosophy from the Bible to postmodernism. â€œThe women of the city are not going to rest until they have secured this public library.â€ â€”Mrs.
Author: Daniel H. Frank
Publisher: Psychology Press
A Chomprehensive anthology of classic writings on Jewish philosophy from the Bible to postmodernism.

domain: library


text:
rewardgold = 500000rewardexp = 500000rewardarmyexp =500000
After the first robber quest is given, save and exit (don't do quest yet)
Go my: my documents\Real Warfare 2 - Teutonic order\profiles\YOUR PROFILE\customs
Find the save quest file. Open with notepad look for the threeitems above (located near bottom of text) change the value to what you would like.(I used 500000)
Exit and save.
Now load the save and complete quest. You will rewarded the value added after you return to your castle and do a transaction.
I recommend the first reward quest It's easy to win.
[Edited by sumidiot, 11/21/2011 12:37:59 PM]
[Edited by sumidiot, 11/21/2011 12:38:40 PM]

domain: game


The Luang Prabang Film Festival has joined forces with Lao New Wave Cinema to launch a series of videos for its newest educational activity, Filmmaking 101. The program consists of nine short tutorial videos for Lao filmmakers in the Lao language, released on YouTube and Facebook as an ongoing reference for creators of media in Laos.
Hosted by Kaewkun Saengphachan (a well-known Lao actor, singer, and filmmaker) and Anysay Keola (president of Lao New Wave Cinema and writer/director of At the Horizon and Above it All), each video focuses on a different aspect of film production. Produced at the Lao New Wave Cinema studio in Vientiane, the videos feature practical shots and detailed explanation regarding numerous skills needed for filmmaking, like how to prepare for a production, ways to achieve certain visual effects, or tips for high-quality editing.
In a country with no film schools and a nascent film industry, the videos are designed specifically for filmmakers with little to no budget in order to show how accessible filmmaking can really be.


Endpoint protection is becoming a real challenge for IT and security professionals, especially in a business environment. While protecting a server or a limited attack surface is usually straightforward, protecting the organization’s endpoints can be complex for a number of reasons.
Most organizations don’t really document the endpoints connected to their network. More importantly, attackers regularly target endpoints for their larger attack surface; after all, endpoints are less monitored compared to intranets and servers.
So, how can sufficient endpoint protection be established? There are four steps you can take to get started.
1. Encrypt All Data
The easiest way to get started with endpoint protection is by encrypting all data. Encryption immediately protects data against unauthorized access because it requires a valid encryption key to be present when the data is accessed. An inability to access the information makes breaching the network pointless.
Data encryption also forces more transparency within the network. When there is a need for a valid encryption key to be present, all endpoints are forced to download the valid encryption key periodically. That process can be used to get a better view of the organization and the devices that team members use.
2. Establish Security Policies
That transparency leads to our second step: the creation of strict security policies. When your organization allows members to bring their own devices, forcing those devices to meet certain requirements before they are granted access to a valid encryption key becomes the easiest way to establish security policies and enforce compliance.
You can also use the opportunity to fully document the devices connected to your network. Not only will you be able to establish and enforce data security policies, but you can do the same with BYOD policies as well.
3. Add Endpoint Protection
An endpoint security system is your best solution in this instance. Rather than relying solely on an antivirus software being capable enough – or having multiple hardware firewall and security suites running within the network – it is much simpler to rely on a holistic endpoint security system.
Endpoint security software suites work really well in this circumstance. You immediately overcome key challenges of securing your endpoints with only one security suite. Management of security measures is easier too.
4. Activate Detection and Prevention
Another good reason why an endpoint security suite is the best tool to use is that it does more than block attacks or minimize the damage. The best solutions integrate machine learning and advanced detection tools to enable endpoint detection and response.
What is endpoint detection and response exactly? As the name suggests, it is the process of detecting zero-hour attacks without relying on existing threat definitions. The use of machine learning certainly helps since it allows the endpoint security suite to respond to potential attacks before those attacks can have a real impact on your endpoints.
By completing these 4 steps, you have a more secure ecosystem and better control over endpoint security. You can continue refining endpoint security by adding extra security measures such as update and device policies, but taking the steps we covered in this article is how you make sure that your endpoints are no longer vulnerable.


text:
I'm house-sitting for a friend who always uses those paper coffee filters set in a little cone shaped holder. These little paper cones must be purchased, and there's always the danger of running out.
I've discovered a super simple method, that takes no more time. The cone shaped holder in the first photo, is made with stainless steel wire mesh that is very fine. I fill it with coffee grounds, and pour water through it. If it's in a small cup, the water must flow through fairly slowly. I use it in a wide cup, that allows the coffee to sit in a bath of hot water.
Here are the steps.
1. Put the desired quantity of grounds into the cone.
2. Set it in the cup.
3. Pour water over the grounds and continue until the cup reaches the desired level.
4. After allowing it to steep for a short time, lift it out and empty the grounds into the compost. Hold it upside down and flick. Most material will fall into the compost.
5. Rinse the reusable cone.
This takes about the same amount of time as using the disposable cone. There's a little more work involved in dumping the compost and rinsing, but a paper cone does not have to be retrieved and placed. Both methods are quicker than using and cleaning a French press.
I'll describe what I'm doing for single serving coffee; it's stupid cheap and very good coffee but rather fiddly and depends on some unique sourcing.
A lot of people aren't fans of brewed coffee (grounds soaked liked tea). A currently-popular ridiculous alternative is the Keurig "pods" brewer -- a $75 to $250 machine (depending on features) that forces hot water rapidly through an envirommental nightmare of a single-use plastic-and-foil pod ($.75-$2.00) containing coffee and filter. It makes pretty good coffee, expensively and conveniently, if you don't mind the cost and the externalities.
Well, I found a working machine at a garage sale for $3.00.
They sell filter baskets so you can use your own coffee in 'em -- this costs you some of the convenience, but it's still very fast, very good single-serve coffee if you use a fairly fine dark roast. However, the reuseable pod-baskets they sell for thist are as stupidly over-priced as the machines, usually more than $10.00 for a few penniesworth of plastic and mesh.
They ALSO sell tiny paper filters you can use inside the little baskets. These make the coffee slightly better by increasing the brew time slightly, and they drive up the up-front fiddle factor by making it harder to load the coffee, but they make getting grounds out again easier. And the tiny filters compost OK. But the price is stupid, like $7/100 -- contrasting with a stack of vastly-larger filters for an 8-cup drip brewer that costs $1.50 for 250.
It turns out that baskets and filters alike can be bought from China for near-nothing (under two dollars delivered) if you don't mind waiting six weeks for delivery.
So that's my current tinkery futz when I want a really good cup of really cheap coffee: packing grounds into a tiny paper filter in a tiny pod and putting it into some idiot's cast-off nightmare of a device for having been smoothly separated from his money.

domain: hobby


SALEH MEN SHORT KURTA R/N
RM71.91 – RM80.91
Welcome to Kamdar where we strive to provide our costumers an array of products that satisfy demand for our quality and effieciency. Buy our products from Tiktok in a worry-free manner as we guarantee 100% authenticity. Shopp[ing can’t get any better than this, so start today!
- Material: Cotton Linen
- Pattern: Plain
- Slevee Length: Long
- Regular fit
- Available Colors: Yellow, Soft Pink, Brown
Disclaimer: Please note that the colors as shown may vary slightly different due to difference in monitor settings.


text:
Rambus and Renesas Electronics Sign Patent License Agreement
Rambus Inc., one of the world's premier technology licensing companies, announced today it has renewed its patent license agreement with Renesas Electronics Corporation (TSE:6723). Renesas Technology Corp. and NEC Electronics Corporation, merged in April 2010 to form Renesas Electronics. This agreement covers the use of Rambus patented innovations in a broad range of logic integrated circuit (IC) products offered by Renesas Electronics. Specific terms of the agreement are confidential.
"This license with a global leader of the semiconductor industry represents continued validation of the strength of our patent portfolio," said Sharon Holt, senior vice president and general manager of the Semiconductor Business Group at Rambus. "We have enjoyed a long and positive relationship with Renesas Electronics, and we are pleased to renew this important agreement with one of the top five semiconductor companies."
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domain: news


Progress Releases Suite of Data Connectors for SaaS CRM Platforms
Progress has announced the availability of a new set of SaaS CRM connectors as part of the Progress DataDirect Hybrid Data Pipeline. The new enterprise-ready connectors leverage industry-standard SQL (ODBC, JDBC) and REST (OData) access, using prebuilt connectors to access data from third-party analytics and data management tools, as well as other SaaS applications.
The new suite of open standard connectors provide powerful and open data connectivity to SaaS CRM solutions, including Salesforce, SugarCRM, Veeva CRM, Microsoft Dynamics and Oracle Sales Cloud, according to the vendor
ISVs in the analytics and data management space need to integrate with and pull data from a wide range of CRM solutions, and this Progress support for SaaS CRM with open SQL and REST connectors offers them a "great option" to connect standalone tools to CRM data for advanced analytics capabilities, said Dion Picco, general manager, Data Connectivity & Integration, Progress.
The Progress suite of CRM offerings with prebuilt standard connectors provides optimized data connectivity by pushing down operations to the SaaS CRM APIs, and supports the latest versions of those APIs.
In addition, the Progress DataDirect Hybrid Data Pipeline is a data access service that provides secure access to cloud and on-premise data sources for hybrid cloud applications. Through the Hybrid Data Pipeline, the connectivity layer of the CRM suite can now be self-hosted in any cloud or on-premise.
The new connectors are available today. To learn more, go here.


text:
"What to Expect": Marianne McKennett and Virginia Fowkes Discuss Training of Physicians for Underserved Areas
Last Updated on April 16, 2022 by Lee Burnett, DO, FAAFP
On Tuesday, April 15, the second day of the 25th National Conference on Primary Health Care Access, Stanford University’s Virginia Fowkes and Doctor Marianne McKennett of the Sharp Chula Vista Department of Family Medicine will comprise a plenary panel discussing “Issues in the Training of Primary Care Physicians for Rural and Underserved Areas”.
The presentation will follow this year’s Charles E. Odegaard Lecture. The late Doctor Odegaard, who was a Fellow of the National Conferences, had promoted, as president of the University of Washington during the mid-20th century, the idea of universities becoming involved in the problems of their surrounding communities.
Dr McKennett, for the past 15 years, has been involved in the training of family physician residents at the Scripps Health Center Chula Vista (California) family medicine residency program. Much of their training occurs in the San Ysidro Health Center, a large federally-qualified health center located near the California-Mexico border in San Diego’s South Bay. (Dr McKennett is a Fellow of the Coastal Research Group.)
Virginia Fowkes, FNP, MHS, Emerita of the Stanford Univesity School of Medicine faculty.
She has been engaged in planning two projects with the California State Area Health Education Center (AHEC), to address the issue of physician burnout in providing medical services in community health centers located in underserved areas.
The California Statewide AHEC is a consortium of non-profit health-related entities located in both rural and urban areas of the State of California. Ms Fowkes is a Senior Fellow of the Coastal Research Group.
Ms Fowkes’ activities with AHEC are focused on retention of clinicians, especially physicians, in CHCs. She observes that many resources have gone into recruitment and training to get clinicians to the underserved areas, but little has been done to keep them there.
Twelve medical directors recently left one of our Los Angeles consortia clinics, ostensibly because of EMRs and dissatisfaction with their jobs.
Nationwide the burnout rate for primary care physicians has risen from 25-46% and it is greater in settings caring for large minority populations. The California AHEC ihas initiated two proejcts to address this serious and growing issue,
The 25th National Conference on Primary Health Care Access will be held April 14-16, 2014 at the Hyatt Regency San Francisco.


text:
There is so much to take in when you walk through the brick wall into The Wizarding World of Harry Potter – Diagon Alley. My little witches and wizards were ready to start making magic as soon as we got there. Why? Because Universal Orlando Resort had invited us down to experience one of the hidden gems—Shutterbutton’s Photography Studio.
The staff welcomed us and explained how we could jump into some of the most iconic scenes from the Harry Potter movies and create our own moving photographs. We were sold and got right down to making magic!
We were fitted with house robes of our choice and even used our own wands as props. (Any item you’d like to bring and use is welcome as the robes are the only props available to borrow.)
We all stood behind a private green screen and were given easy directions to shoot our big screen debut. These moving photographs included 12 iconic scenes from The Wizarding World of Harry Potter – Diagon Alley and Hogsmeade.
We screamed in terror from the fire-breathing dragon on Gringotts Bank, and my son’s favorite scene was having his little sister appear on the “Have You Seen This Wizard?” poster. After we got to see a short preview of our scenes, they provided us with a DVD to take home, along with a digital copy.
The whole experience took about 20 minutes and we left with what I’m calling “the coolest souvenir ever”. It’s earned its place next to our wedding video, and I’m sure we will watch, laugh and remember our trip to The Wizarding World of Harry Potter for years to come.
The kids also love showing it to every friend that has come over, laughing each time they see themselves. Their friends think it’s pretty cool, too!
I would definitely make sure to stop into Shutterbutton’s before you leave Diagon Alley. These are the memories that become etched in time, as experiences like these are ones we will never forget.
Siera is a travel agent with Destinations in Florida, Universal Studios Travel Agency of the Year 2010, 2011, & 2012. She would love to help you plan your next Universal Orlando vacation at no cost to you. Contact Siera today at email@example.com or at 734-771-1290 for more information or your free, no obligation quote.