text:
DJI’s Mavic Pro made waves for its transportability: It was the initial drone to have arms that folded up against its body, making it supercompact. Since it launched, lots of others have fielded duplicates with a similar design, however it looks like only DJI could knock the Mavic Pro off its perch as our favorite electronic camera drone. As well as it has, with the Mavic Air.
Smaller (as well as $200 less) compared to the Pro, the $799 Air also has folding arms as well as the same 4K camera and gimbal as the Pro. But the most up to date design likewise features a brand-new object-avoidance mode that makes it even less complicated to pilot in narrow spaces. It’s the best camera drone yet.
It’s like the Mavic Pro, just smaller sized. The Mavic Air has a sleek appearance with jumping curves that make it appear like it’s flying, also when it’s on the ground. Like the Pro, the Air’s 4 arms rotate exterior from the text for trip mode.
When folded, the Mavic Air is about 3.5 inches vast and 6.5 inches long. With the arms extended, the drone expands to about 7 inches vast, not including its rotors.
The Mavic Air has a smooth look with swooping curves that make it resemble it’s flying, even when it gets on the ground.
The front of the drone houses its 4K cam as well as three-axis gimbal, which coincides as on the more expensive Mavic Pro. Here, though, the video camera is secured on two sides by the drone’s body. On the Pro, the video camera was more subjected, so a clear plastic shroud was had to shield it during transport. Mavic Pro In Stock
The Air is studded with sensing units that help it browse its atmosphere: Over the primary camera are two forward-looking sensing units, while the underside and also back add 2 even more sensing units each.
The bulk of the bottom of the Air is occupied by its removable battery, which has 4 lights to show just how much fee it has.
Much like the controller for the DJI Spark, the Mavic Air’s controller has two arms that extend from the main body to hold your mobile phone in place. I such as that the Air’s controller utilizes a physical cable television to connect it with your phone (there are adapters for Android as well as iOS gadgets); with the Spark, you had to very first set your phone wirelessly with the controller, and then with the drone– a minor however frustrating extra step.
Or else, the Air’s controller is much the same: Two joysticks regulate altitude and activity, while shoulder switches revolve the camera, take pictures as well as video clip. Other switches on the face land the Air, engage Sport mode and power the controller on and also off. Every little thing is outlined well. Any individual that’s made use of a PS4 or Xbox controller in the last decade will certainly have not a problem figuring out ways to utilize the Air’s version.
An additional wonderful touch: The controller sticks for the Air unscrew and also could be stowed in the body of controller, that makes it much easier to carry around. I seemed to always forget this, however, and needed to unfold the controller’s arms, store the sticks and also fold the arms back up once again.
If you wish to miss the controller entirely, the Mavic Air, like the Spark, could be controlled using nothing greater than hand motions. You can launch the Air, move it up, down, right as well as left, and also take pictures as well as video clips.
These motions all functioned relatively well, yet in some cases, I would need to attempt a couple of times before the Air’s cam would certainly acknowledge my hand motion. The Air’s variety is likewise limited to about 20 feet in this setting, since the camera should have a clear sight of your palm. Mavic Pro In Stock
Similar to all DJI’s drones, the Mavic Air was an incredibly easy drone to fly. It hovers well in all yet really solid wind gusts (the app will certainly advise you if it’s too windy) as well as zips around easily.
A brand-new function called Advanced Pilot Assistance Systems (APAS) will cause the Air to immediately fly about things, such as trees and also shrubs. When that feature is triggered, the Air flies a little bit slower than typical, however APAS functions well. I found it was particularly useful when I flew the drone via wooded locations. Nonetheless, the Air doesn’t go back to its initial program after dodging a things; you’ll need to reorient it manually.
In Sporting activity mode, the Mavic Air moves. According to DJI, it could strike rates of as much as 48 miles each hour. While I do not have a radar weapon, the drone covered the approximate size of a football area in about 3 seconds, and sounded like a bat from heck doing so.
Video and also Image Quality
As it has the same 4K video camera and three-axis gimbal as the much more costly Mavic Pro, video clips as well as pictures taken with the Mavic Air were excellent.
The cam did an excellent task adapting to various illumination conditions. In one scenario, I introduced the drone at dusk, facing the setting sun. As it rose right into the air, the Mavic Air was able to maintain your homes in the darker foreground properly lit, while still showing off the glowing hues of the sunlight. I did notice a little noise in the darker areas, yet that’s to be anticipated.
On a clear wintertime’s day, the Mavic Air’s video camera was sharp sufficient to make sure that I can make out the high-rises in Manhattan, about 15 miles away.
Saving 4K video clips takes a little bit longer compared to lower-res files. After I stopped a recording, the Air would certainly commonly take about 10 to 15 secs to finish refining what it had just videotaped.
In HDR mode, the Mavic Air can take a series of three photos at different direct exposures and also incorporate them into a single shot. I likewise such as that the drone shops each photo separately, so you could use a picture editor to modify the HDR yourself.
It came to be a bit irritating to dig via food selections on DJI’s application to locate things such as the view or round mode for the Mavic Air cam.
Just like the Spark, the Air has a couple of autonomous trip settings, called QuickShots, that allow you capture some very remarkable video clip. The two latest, Asteroid and Boomerang, are quite wild; Asteroid has the drone fly as much as a pre-programmed altitude as well as take a 360-degree photo. And afterwards it incorporates that with a video as it flies down to you. Mavic Pro In Stock
Through the DJI app, you obtain pretty granular controls over the electronic camera, letting you adjust direct exposure, shutter rate as well as more. Nonetheless, it became a little bit annoying to dig with food selections to locate things such as the scenic view or spherical mode for the electronic camera.
An additional nitpick: When I switched from video clip to picture mode, the app’s interface resized and diminished to represent the various element ratio of still shots. Nonetheless, this likewise compressed every one of the on-screen controls.
DJI says that the Mavic Air should stay up for around 20 mins, and I discovered that to be close to accurate. That’s only a few minutes much less compared to the Mavic Pro, as well as absolutely a practical trade-off, given the reduced rate of the Air. The drone ships with a single battery, which could conveniently be exchanged out; extras set you back $79 each.
By itself, the Mavic Air costs $799, and also is available in white, black or red. It consists of a controller, two sets of propellers, prop guards, a charger for a solitary battery and also a case for the drone itself.
The Fly More Combo ($ 999) consists of the controller, three sets of propellers, three batteries, a battery charger that can fit four batteries, plus a messenger bag-style carrying instance that can accomodate the drone, batteries and also controller, as well as an extra collection of props. The bag itself is extremely appealing and also made it really hassle-free to tote whatever.
A 3rd package, which cost $1,299, consists of every little thing in the Fly More bundle and also adds in a collection of DJI safety glasses, for a first-person view from the drone.
When the Mavic Pro appeared, I believed DJI had actually made the most effective drone ever. With the Mavic Air, it’s again made a drone that’s the envy of all others. The Mavic Air is mobile, maneuverable, takes great photos and video clip, as well as comes loaded with a variety of goodies, such as the automatic camera modes and item avoidance. At $799, it’s $200 less compared to the Mavic Pro, yet uses nearly all of the exact same features. (I ‘d spring for the $999 Fly More package to obtain 2 added batteries, the lugging case and also the battery charger.) If you’re seeking the most effective well-rounded consumer drone, the Mavic Air will fly you right.
Click here to purchase your personal DJI Mavic Air. Mavic Pro In Stock


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text:
Grin with Grace. Do you know what that is? I knew it was the title of Kathy Carlton Willis’s new bible study book but that’s about it. I didn’t know it would come to me at a time when I really, I mean, REALLY needed to read it.
What grinning with grace meant to me was something really personal. I had been dealing with a co-worker that I just don’t see eye to eye with. One day she came to me and asked if I was mad at her. My initial thought was “yes, I’d love to rip your face off and hand it to you”. I had been reading Kathy’s book and this woman’s face kept flashing before my eyes. I stopped and prayed before I spoke. I even said to her “I’d better pray before I speak so that God can come out. Otherwise, the world will come out of my mouth and we know the world is a vicious place.”
I asked for grace and wisdom because I remembered one passage from Grin with Grace very vividly:
“So here’s what I want you to do, God helping you: Take your everyday, ordinary life – your sleeping, eating, going-to-work, and walking-around life – and place it before God as an offering. Embracing what God does for you is the best thing you can do for him. Don’t become so well-adjusted to your culture that you fit into it without even thinking. Instead, fix your attention on God. You’ll be changed from the inside out. Readily recognize what he wants from you, and quickly respond to it. Unlike the culture around you, always dragging you down to its level of immaturity, God brings the best out of you, develops well-formed maturity in you.
~Kathy Carlton Willis, Grin with Grace, page 21.
My co-worker and I were able to talk about our issues. I was able to own up to my part with grace and we both left that day in a much better place. I don’t know about her but I know I grinned with grace all through that conversation, and I know if I had not read Grin with Grace I probably would still be ready to rip faces off.
More about Grin with Grace
Each chapter features five sections:
· Grin with Grace contains real-life stories and observations. You’ll laugh at the humorous confessions and wacky insights, and relate to Kathy’s transparent honesty. Be inspired to see grace in your everyday life.
· Grow with Grace features a grace word study. Kathy examines Bible verses and personalizes the meaning to your situation. Workbook prompts allows you to write down your thoughts as you read along.
· Go with Grace offers life application. Pick one action step and make it work for you, or pick all of them—but do something to live out your grace-walk.
· Give with Grace advances life application further, equipping you to become an instrument of God’s grace to others. You’ll be inspired to take what you’ve learned and give it away to others. This is when faith becomes ministry—when your focus expands to see the needs of others.
· Your Grin with Grace Challenge describes a grace-challenging scenario to give you an opportunity to exercise your newfound grace. It allows for speculation and judgment calls, to prepare you for the what-ifs that happen in life.
Release Date: May 1, 2015
Praise for the Book:
Grin with Grace is perfect for women on the run, since there is no set daily reading schedule. The blend of humor, transparency, biblical concepts, word studies and opportunities to plug what I learned into my life gave me a fresh approach to doing my devotions or reading a Bible study.
Dr. Thelma Wells (Mama T)
President of A Woman of God Ministries
CEO, That A Girl & Friends Speakers Agency Speaker, Author
Kathy Carlton Willis writes and speaks with a balance of funny and faith—whimsy and wisdom. Not many funny girls also have Bible degrees! Kathy’s a pastor’s wife, which gives her plenty of opportunities to grin with grace. She shines the light on issues that hold women back and inspires their own lightbulb moments. Almost a thousand of Kathy’s articles have been published in books, magazines, newspapers and online publications. Kathy’s tagline describes her best: Light & Lively: His Reflection/Her Laughter. Kathy lives with her pastor/husband, Russ, in Rockdale, Texas.
Where can you find Kathy:

domain: literature


text:
Ryan Fuhrmann (RSS)
- 7 Healthcare Stocks to Buy Now
- Qualcomm, Inc. (QCOM) Stock Should Dominate the Mobile Phone Chip Realm Indefinitely
- Sears Holdings Corp (SHLD) Stock Is Garbage, But Should You Short?
- Freeport-McMoRan Inc (FCX) Stock Doesn’t Have What It Takes
- Twilio Inc (TWLO) Stock Is in an Uber-Competitive Space

domain: economics


text:
Vacation rentals in El Armadillo
Find and book unique accommodations on Airbnb
Top-rated vacation rentals in El Armadillo
Guests agree: these stays are highly rated for location, cleanliness, and more.
Condo in Moroleón
Comfortable apartment for short to long stays
Enjoy the comfort of this quiet and central accommodation. There are also several convenience stores near the home, restaurant, and other food services, as well as supermarkets in case they require pantries. It is also close by for shopping days in the metropolitan textile plaza, the clothing stores from October 12, and others. Great for cutting and long stays.
$32 per night
Cabin in Valle de Santiago
CABAÑAS RUIZEÑOR "La michoacana"
In Ruizeñor cabins we have 3 cabins for different tastes and capacities. We offer: Cabaña La coqueta (wooden) Cabaña La Alfonsina (adobe ) Cabaña La michoacana (rustic wood) "La michoacana" is a nice and cozy cabin for 4 people, located inside a crater, with beautiful views and common outdoor areas. To see our other cabins click on my profile.
$105 per night
Vacation rentals for every style
Get the amount of space that is right for you

domain: travel


Published in International Journal of Manufacturing Technology and Management, Volume 17, Issue 1/2, January 1, 2009, pages 82-102.
NOTE: At the time of publication, the author Ahmed Deif was not yet affiliated with Cal Poly.
This research is concerned with studying the dynamic performance of reconfigurable Manufacturing Planning and Control (MPC) systems. Such goal requires two main tasks. The first task is to develop a dynamic MPC system model that has the ability to reconfigure to different MPC policies. The second task is to design a supervisory control unit that has as input the high level strategic market decisions and constraints together with a feedback of the current manufacturing system state and then select the optimal suitable operation mode or policy at these conditions. This paper addresses the first task of the proposed research and presents and analyses a dynamic reconfigurable MPC model. The response of the developed model to sudden demand changes under different parameters settings is analyzed. In addition, the stability limits of the system are also studied. The results give a better understanding of the dynamics of reconfigurable MPC systems and the different trade-off decisions required when selecting an MPC policy and the limits for parameters settings. These results represent the first step towards designing the supervisory control unit which will be responsible for managing the reconfiguration of the whole system.


text:
Best Quilt Shop In Bird in Hand
Martha & Ruth were the salesladys that helped us with our selection of a beautiful quilt to fit our Amish bed which when it arrived at our Az home matched perfectly. They were knowledable and very helpful. My wife and I enjoyed the experience and would recommend the shop to anyone!!
They offer a full array of bullion products as well as coins. Their prices are less than all of the other places I have shopped at online. I had to wait in line, these guys were that busy.
my favorite bead store!
Buttercup Beads is my favorite bead store - they have the best items and the most varied classes. The staff is really helpful and knows about all the jewelry making techniques. It is a really fun and beautiful store and they also have Chamilia Beads, stones, czech crystal, swarovski, seed beads, instructions, and kits. website is great www.buttercupbeads.com
Best Train Shop and I'm Very Picky
Great stock, pleasant service, didn't feel rushed, competetive prices. Worth the Trip even from Hunterdon County!
Buss paint is the finest paint store I've ever shopped in. The guys in the paint dept. are super knowledgeable. and polite. The stain they matched f...

domain: hobby


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Just a quick share today. A little while ago I had an urge to make something small. Something that I could finish super quick. So I turned to Lucy at Attic24 - and her beautiful triple layer flower.
I made one in record time and decided it would make a lovely gift for a friend, and in my true scatty style I packed it off in the post before I even took a photo!!
So last week I made 2 more, one for me and one for Miss M, and we both love them!!
And the sun even shone for some photos :)
Mine is brightening up my coat now, very pretty!
I also wanted to let you all know that Henry's Shed has a facebook page!
I set it up a while ago but it has been a little neglected. I am going to try super-hard to use it more often (as it is sooo much quicker than blogger!)
It would be lovely if you could pop over and visit me and maybe give my page a like!?!?
See you soon!

domain: artistic


Every great journey begins with the decision to take the first step. The first step is sometimes the hardest but you must do it. You were not placed on this earth to be stay hidden by fear, you were placed here to pursue and achieve great things.
It’s time to kick it up a level.
Preshrunk fleece knit minimize shrinkage during use to always give you that perfect fit
Double-lined hood with color-matched drawcords completes the polished, aesthetic look.
Air jet yarns to create a soft-feeling, anti-pilling pullover that guarantees comfiness during extended use
Double-needle stitching at waistband and cuffs for improved durability


tHE PERFECT PISTOL SHOT BLOG
Marksmanship is best learned as a study; an intellectual process that enables the shooter to train for consistent results. It is extremely difficult to teach a student concepts on the firing line, and yet most instructors spend very little time in the classroom. Law enforcement is the greatest offender of presenting poor continuing firearm education. That is why so many officers consider qualification to be a matter of luck.
Albert League is a former Marine Corps and law enforcement firearm instructor who consults on a variety of security topics. He is the founder of the Practics firearms defense system and author of the Practics book series.(www.practicsusa.com)


text:
Prong Set Diamond Chain EarringEC4183-R
This isn't your traditional earring. This fascinating beauty lays perfectly on your ear, shining in 14k gold and diamonds alternating on the chain.
Check out the details...
- 2.1G high polished 14k gold
- 0.33 ct. tw.
- Finished with a butterfly backing
- Sold individually

domain: design


text:
Natural Zeolite For Agriculture
Zeolite has a high Cation Exchange Capacity (CEC) that enables a greater loading of plant nutrients such as nitrogen & micronutrients, contributing also to the improvement of fertilizer efficiency. Because of its high bonding capacity for ammonium, potassium and other ions, clinoptilolite is the one natural zeolite that is used mainly in agriculture.
Zeolite is has a wide range of benefits including:
1. Improve the efficiency and value of fertilizer for betterplant growth
2. Improves water based soil retention
3. Natural zeolite helps avoid fetilizer loss and reducenurtients loss in the soil
4. Improves water infiltration and retention
5. Improves long term soil quality
Zeolite can also be modified to make slow release fertilisers and can be used as support for herbicides, pesticides or micro-organisms which promote healthy plant growth.

domain: chemistry


text:
I'm a little bit obsessed with the "Pirates of the Caribbean" OPI nail polish line. It's beautiful. Absolutely beautiful. The other day, I bought "Sparrow Me the Drama" and "Steady as She Rose." They are both so dainty, pretty and tropical. That's kind of a wonderful combination. Anyway, because it's a special collection, it is only here for a limited time, so I suggest you go out and buy all of the shades. Perfection.
Aren't they all so pretty?! I kind of want the purple or mint green colors too. Ugh, I have an illness, I want all of them.
Sparrow Me the Drama (This is my favorite one).
Steady as She Rose (I also love this one too. Gosh, I am never satisfied, am I?)


kothariya, Rajkot, Gujarat
GST No. 24BJVPP4317E1Z1
Approx. Rs 30 / FeetGet Latest PriceProduct Details:
|Minimum Order Quantity||100 Feet|
|Finish||White Powder Coated|
|Capacity||50 kg per bracket|
|Packaging Type||10 pcs per box|
|Surface Finishing||Powder Coated|
|Carton Box||10 box per cartoon|
|Country of Origin||Made in India|
Iron material Cotting surface & Heavy-duty channel bracket.
our heavy-duty product channel 1.5mm thickness & bracket 1.2mm thickness.
the best quality product of channel & bracket.
we also supply ss Clip & vacuum for bed of bracket.
our product double slotted for strength.
We deal in the best quality product. It is a 6" to 20' Bracket. It has a It has a White Powder coated. It can be used for shop and for storage purposes and the weight is around 1 kg for the 6 inch channel. Sizes are 6' & 8' for channel & 6" to 20" for Bracket
Explore More Products


text:
Chicory with anchovies
The slightly bitter taste of chicory and salty anchovies combined nicely into such a delicious side dish.
- Peeled chicory, wash well and chop.
- Put in a pot of boiling water and boil briefly.
- Wash the anchovies under water and chop them finely.
- Fry the unpeeled clove of garlic in olive oil, add the anchovies and stir until well decomposed.
- Stir in the chicory, season with salt and pepper and cook for another 5 minutes.

domain: food


text:
Dancing at the Asylum, Marilyn Ricci
Quirky Press, 2021 £5.00
Blurring the genres
Dancing at the Asylum tells the story of Carlton Hayes Hospital in South Leicestershire, originally Leicestershire and Rutland Lunatic Asylum. The asylum is real, and the book is dedicated to those who died at the asylum between 1908 and 1994, buried in an unmarked mass grave.
Throughout, the pamphlet blurs genres in several ways. Firstly, although it is formatted in lines and stanzas resembling poetry, it functions in a way that’s similar to a novel. This work is highly narrative and serves to tell two characters’ stories — we learn how Edward was traumatised fighting in the Second World War, and how Mary has had multiple pregnancies out of wedlock, resulting in miscarriage or adoption.
The language is undoubtedly poetic — take Edward’s description of one of his comrades:
Trooper Doyle’s face was a freckle
under a red shock of hair, blue orbs
circled in white desert dust.
However, the ultimate purpose of the poetry remains to tell a story, and to characterise Edward and Mary. Ideas of sanity and insanity, and how we treat those whom society deems mentally ill, are explored not through abstract imagery but through human tales.
At times, this pamphlet also seems to cross over from poetry into script, with moments of dialogue acting to move the story forward. For example, we learn through dialogue that (on admittance to the asylum) Mary is pregnant, and not for the first time:
When did you last have relations, Mary?
Aunt Jane came with a cake
No, a man. [...]
You’re three months gone.
This snippet of conversation conveys the facts of Mary’s situation at the same time as revealing her vulnerability and naiveté.
Perhaps the most significant way in which Dancing at the Asylum blurs boundaries is when it comes to fact and fiction. The setting’s a real one, but the characters are fictionalised. Despite this, the work paints a believable picture of these two individuals, even supplying admittance reports resembling facsimiles of real hospital documents.
Edward and Mary push against neat boundaries of sanity and insanity, presenting as complex, full human beings with rich histories. So it’s fitting that their stories be told in a way which is, in itself, innovative and genre-bending.
Creating a marker
Few pamphlets have as moving a dedication as this one. It is dedicated to
the 1,150 people who, between 1908 and 1994, died in Carlton Hayes Hospital […] and were buried nearby in a mass grave in Narborough cemetery without headstones or markers of any kind.
So the publication itself creates a kind of marker for the dead, a way of honouring them. Carlton Hayes was what we once called ‘a lunatic asylum’. And it had nearly twelve hundred people in its mass grave. What an utterly chilling thought!
But the particular detail that fired Marilyn Ricci’s imagination was the fact that the hospital also had ‘the best dance floor in the country’ — a light in much darkness, the thought of those dances.
And so she creates two fictional inmates, one (Edward) admitted in 1947 and the other (Mary) in 1966. In dramatic monologues, one for each character, she brings them alive, allows them to meet, and finally leaves them quick-stepping on the dance floor.
And the two characters do come alive. Edward, during active service in the Second World War, has been traumatised by an explosion. He’s haunted by the memories:
A long piercing whistle, we gag on oil, a flash
of green, amber and red. Marsh stares at the place
his hand used to be. Doyle lies in his seat
as though he’s asleep.
In the hospital, Edward sees Mary and recognises a particular ‘look on her face’. They have nothing in common, and everything in common. ‘Mary, don’t hold your grief like a prize’, he thinks.
Dancing at the Asylum is a beautiful piece of dramatisation. It begs for a radio broadcast where the characters speak while the listening imagination fills out the scene.
Edward comes from a classy background. Mary is made of more ordinary stuff. She doesn’t have his education, his references, his elegant choice of words.
But she has a moment of magic, and it’s transformative; it’s her marker. I won’t forget her:
Her hands feel like clouds soft with rain and, boy,
can she dance! Feather-footed in her scarlet dress,
rose in her hair and nothing like Marjorie on the


text:
Trip Planner: Asia / India / West Bengal / Darjeeling District / Darjeeling / Himalayan Tibet Museum
Learn about Tibetan culture and history at Himalayan Tibet Museum. Situated within walking distance from the town center, the museum was opened in 2015 on the ground floor of a non-profit organization for preservation of Tibetan heritage. In addition to Tibetan cloth paintings, traditional musical instruments, prayer bells, and other artifacts, the museum also houses a moving photo exhibit depicting the exodus of Tibetan refugees. Some of highlights include a 3D model of the Himalayan plateau and a replica of Potala Palace, the former seat of Tibetan government. Choose to start, finish, or center your holiday on a trip to Himalayan Tibet Museum by using our Darjeeling trip maker.
Himalayan Tibet Museum reviews
This is an must visit place to learn more about the history, language & its culture of Tibetians.... more »
Whilst it's not particularly large, this has to be one of the most illuminating and loving museums I have visited. The displays are beautifully crafted, and the adoration and respect of the current... more »
This is a government run museum and is opened from 10 am in the morning. It is good. Rates for children are Rs 25 and adult Rs 50. The location of the museum is in a narrow lane and there is no parking space available for vehicles. It is visible while driving the vehicle. The museum is located near Clock Tower.
Very peaceful and knowledgeable environment, with professional staff. Service provided by the staff is very well and good.
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Dr. Barry M. Blechman is security consultant and an expert on arms control and national security. He is the co-founder of the Washington based ‘security’ think-tank the Henry L. Stimson Center, and the founder of DFI International, a Washington based research and consulting company sold in 2007.
Education and Career
Blechman has a Masters Degree from New York University and a PhD in international relations from Georgetown University. He has taught at the Johns Hopkins University, Georgetown University, and the University of Michigan.
He was Assistant Director of the Arms Control and Disarmament Agency from 1977 to 1980. He served on the Commission to Assess the Ballistic Missile Threat to the United States (The Rumsfeld Commission) (1998-99), the Defense Policy Board (2002-06), and the Mayor’s Bioterrorism Preparedness and Response Program Advisory Committee in the District of Columbia (2004-06). He has served as assistant director of the U.S. Arms Control and Disarmament Agency, directed the defense analysis staff at the Brookings Institution.
- Barry M Blechman, Stephen S Kaplan, et al., Force without war: U.S. armed forces as a political instrument (Washington, Brookings Institution, 1978), (ISBN: 0815709862 9780815709862; 0815709854; 9780815709855)
- Barry M Blechman ed., Rethinking the U.S. Strategic Posture: A Report from the Aspen Consortium on Arms Control and Security Issues (Cambridge: Ballinger, 1982)
- Barry M Blechman, Preventing Nuclear War: A Realistic Approach, (Bloomington, Indiana University Press, 1985), (ISBN: 0253203503)
- National Defense University
- Committee on the Present Danger
- Brookings Institution
- Advisory Committee on Transformational Diplomacy, Member
- Detica Group plc, senior consultant
- Barry M. Blechman and Janne E. Nolan, ‘Reorganizing for More Effective Arms Negotiations’, Foreign Affairs, Summer 1983
- Henry L. Stimson Center, About (accessed 16 May 2008
- National Defense University, Barry M. Blechman, (accessed 16 May 2008


text:
The right to dissent is what has made America great, says Reuben Guttman.
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The path to leadership change is fraught with challenges - even for McKinsey - but, says management guru Patrick McKenna, there are some obvious pitfalls to avoid.
Posted: 09 November 2017 | Management speak
Spanish law firm Garrigues is developing its version of a Spanish speaking artificially intelligent robot, says CIO César Mejías.
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When a firm leader's departure is predictable, firms need to take appropriate steps to ensure a controlled and effective succession process minimising the inevitable 'disruption' likely to occur.
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domain: news


text:
Women's Struggle for Oppression in the Cantik Itu Luka Novel by Eka Kurniawan
Keywords:Struggle, women, oppression, feminism.
This study aims to describe the form of struggle of the main character Dewi Ayu in the novel Cantik Itu Luka by Eka Kurniawan. In the novel, Dewi Ayu fights against the oppression of women and the steadfastness of the main character fighting for women's rights. In society in general, gender differences make women looked down upon by men. The problem in this study is How did the oppression of women occur in the post-colonial period according to the novel Cantik Itu Luka By Eka Kurniawan? What is the position of women, especially those who are still young in the novel Beautiful That Wound?, and How is the resistance of women in fighting for their rights?. In this study using the approach of the flow of feminism. Feminism is an effort to equalize the position between women and men. This type of research is descriptive qualitative.
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This work is licensed under a Creative Commons Attribution 4.0 International License.

domain: library


text:
Characterization of disease-associated proteins improves our understanding of disease pathophysiology. Obtaining a comprehensive coverage of the proteome is challenging, mainly due to limited statistical power and an inability to verify hundreds of putative biomarkers. In an effort to address these issues, we investigated the value of parallel analysis of compartment-specific proteomes with an assessment of findings by cross-strategy and cross-omics (proteomics-transcriptomics) agreement. The validity of the individual datasets and of a “verified” dataset based on cross-strategy/omics agreement was defined following their comparison with published literature. The proteomic analysis of the cell extract, Endoplasmic Reticulum/Golgi apparatus and conditioned medium of T24 vs. its metastatic subclone T24M bladder cancer cells allowed the identification of 253, 217 and 256 significant changes, respectively. Integration of these findings with transcriptomics resulted in 253 “verified” proteins based on the agreement of at least 2 strategies. This approach revealed findings of higher validity, as supported by a higher level of agreement in the literature data than those of individual datasets. As an example, the coverage and shortlisting of targets in the IL-8 signalling pathway are discussed. Collectively, an integrative analysis appears a safer way to evaluate -omics datasets and ultimately generate models from valid observations.
High-resolution –omics technologies hold the promise of significantly improving our knowledge of disease pathophysiology. Integration of –omics data and their in-depth interpretation in the context of the existing literature, are required to maximize the knowledge extracted from individual datasets. Implementation of this approach could catalyze the development of novel biology-driven drug targets1. In particular, studies at the protein level are highly relevant, since proteins directly reflect the disease related phenotypic changes and comprise the vast majority of approved drug targets2,3. Although recent advances in mass spectrometry (MS)-based technologies enable proteomics investigations with increased sensitivity, numerous challenges remain to be met, mainly related to the proteome vast complexity and (biological) variability, mandating the analysis of multiple independent samples in order to reach statistical significance4,5. Additionally, to increase proteome coverage, extensive fractionation at the peptide and/ or protein level have been advocated6,7,8. The latter include enrichment strategies for secreted proteins, which have gained increasing attention, as means to understand cancer invasion9,10,11.
Regardless the applied technique, proteomics analysis generally delivers numerous potentially disease-associated proteins. This is especially of value in Systems Biology approaches12,13,14,15,16 targeting to obtain a spherical view of the disease molecular profile and underlying causative events, and where comprehensiveness is needed. However, verifying all of the identified changes at a single protein level, e.g. via immunohistochemistry or ELISA, appears an impossible task, hence frequently compromising validity of the vast majority of reported –omics findings. To increase confidence on the results obtained from large-scale experiments, an integration of various –omics datasets appears to be a valuable alternative17,18. In the study presented here, we investigated if cross-omics comparisons and respective investigation of consistency in trends of expression are in fact increasing the validity of the obtained results. In addition, and specifically for proteomics investigations, we target to show that the application of different fractionation strategies, - besides increasing confidence in individual findings via cross-strategy agreement,- increases proteome coverage and facilitates shortlisting of biologically relevant biomarkers.
As a model system, we chose metastatic bladder cancer (BCa) represented by two syngeneic cell lines, T24 and its metastatic subclone T24M. Metastatic BCa is associated with very low survival19, hence, understanding the molecular processes and identifying improved therapeutic targets is an unmet, clinical need. High-resolution LC-MS/MS analysis was conducted on samples enriched in secreted proteins, (isolated from conditioned medium-CM and Endoplasmic reticulum and Golgi apparatus (ER/Golgi) fractions, as carrying the cargo of secreted proteins), as well as total cell extract (CE). Total RNA sequencing analysis was utilized to complement and validate the large scale proteomic data sets. To assess the validity of findings in an unbiased way, these findings were compared to literature data represented by the BcCluster BCa database (http://bccluster.org/)20 and retrieved using the GLAD4U tool (http://bioinfo.vanderbilt.edu/glad4u/)21. As shown, cross-strategy and –omics comparisons at the individual molecule and pathway levels increase the credibility of individual observations and improve proteome coverage consequently increasing data extraction from individual –omics experiments for further systems biology and/or targeted investigation.
Proteomic data assessment
The high-resolution proteomic analysis was performed on samples enriched in secreted proteins (analysis of CM and ER/Golgi fractions) and CE, aiming at increasing proteome coverage. The respective workflow is depicted in Fig. 1. The results from 5 independent experiments per cell compartment indicate high-resolution and good reproducibility of the applied procedures. As shown in Table 1, for each experimental approach an average ( ± SD) of 10,062 ( ± 466), 7,298 ( ± 490), 6,053 ( ± 1,407) peptides, corresponding to 1,944 ( ± 85), 1,515 ( ± 75), 1,116 ( ± 164) proteins were identified in CE, ER/Golgi and CM, respectively. Detailed lists of proteins identified per individual MS-run (including/ excluding single peptide IDs) are provided in Supplementary Table S1. To increase reliability of protein identification and differential expression analysis, only proteins identified based on at least 2 unique peptides (in each individual run) and in at least 3/5 replicates in each cell line were considered for further analysis. The reproducibility rates were high with overlap among replicates on average of 77% (CE), 73% (ER/Golgi) and 76% (CM) of proteins detected in at least 3/5 replicates in each case (Supplementary Fig. S1). These corresponded to a total number of 1,359, 1,062 and 816 non-redundant proteins from CE, ER/Golgi and CM, respectively, considered for further differential expression analysis (Supplementary Dataset S1).
To obtain an estimate of the enrichment efficiency for secreted proteins, the SignalP algorithm, which predicts the presence of signal peptides (defining “secreted” proteins), was employed22 (Supplementary Dataset S1). In overall, 30% of proteins in CM were predicted to have signal peptide in comparison to 14% in the ER/Golgi fraction and 9% in CE, indicating the relative efficiency of the enrichment strategies. The normalized signal intensity of these ‘secreted’ proteins corresponded on average to 49.80%, 16.72% and 6.67% of the total intensity for CM, ER/Golgi and CE, respectively. Moreover, enrichment efficiency was assessed based on the normalized average intensity values of specific proteins being representative for each fraction (Supplementary Fig. S2). Actin cytoplasmic 1 and histone H2B type 1-K (protein markers for CE) were highly expressed in CE, whereas their abundance was reduced in CM (by approximately 2 fold for Actin, 10 fold for H2B type 1-K), and for H2B type 1K also reduced (approximately 4 fold) in ER/Golgi (Supplementary Fig. S2). On a similar note, calumenin and 78kDa glucose regulated protein (markers for ER/Golgi) levels were higher in ER/Golgi (by about 2 fold) compared to CE and CM. Cathepsin B and Proactivator polypeptide (markers for CM) levels were found increased (by at least 5 fold) in CM compared to CE and ER/Golgi (Supplementary Fig. S2). Taken together, these results support that the different strategies provided to some extent complementary information. However, large overlaps could also be observed (described below) allowing for investigation of consistencies among the differentially expressed proteins per method, as a means to increase confidence in individual observations.
Complementarity of proteomic profiles
Comparative analysis of the 1,359 proteins identified in the CE, the 1,062 proteins detected in ER/Golgi and the 816 proteins found in CM revealed an overlap of 498 proteins (Fig. 2). This “core proteome” included multiple enzymes, ribosomal and cytoskeletal proteins, some signalling proteins and also abundant chromosomal proteins (such as histones; Supplementary Dataset S1). Each experimental approach also enabled the identification of multiple proteins not detected by the other two methods (408 for CE, 166 for ER/Golgi and 219 for CM; Fig. 2). The former included various nuclear and transcription factors and mitochondrial enzymes, the ER/Golgi fraction had multiple proteins synthesis-related (Protein Niban, ribosomal proteins, DnaJ homolog subfamily C member etc) and signalling proteins (RAS-related proteins, kinases, cell membrane receptors such as EGFR etc) and the CM fraction included various growth factors, interleukins, matricellular proteins and proteases, indicating a good degree of complementarity between these strategies (Supplementary Dataset S1).
Proteins exhibiting a nominal significant change (p < 0.05, Mann Whitney test) in their expression levels (>1.5 fold change) between the two cell lines in respective subcellular fractions were defined as differentially expressed. Based on these requirements, 253 (144-up and 109-down regulated), 217 (116-up and 101-down regulated) and 256 (169-up and 87-down regulated) proteins were considered as significantly altered among CE, ER/Golgi and CM, respectively in T24M vs. T24 cells (Supplementary Dataset S1). Upon Benjamini-Hochberg correction and considering the adjusted p-value (p < 0.05) and the fold change threshold ( >1.5), a total of 171 and 206 proteins were defined as differentially expressed in CE and CM, respectively (Supplementary Dataset S1); whereas none of the ER/Golgi differentially expressed proteins remained significant upon application of FDR correction. This indicates higher variability of this specific dataset, likely being a consequence of the applied multi-step enrichment protocol. Considering the low number of samples analyzed (n = 5 per group) as well as the observed consistency in expression trends among different fractions (as explained below), we further focused on the differentially expressed proteins (>1.5 fold change) defined using unadjusted p-value.
To obtain an initial insight in the biological function of the observed differentially abundant proteins per approach (i.e. the aforementioned 253, 217 and 256 proteins identified in CE, ER/Golgi fraction and CM analysis, respectively), gene ontology information deposited in protein databases (Uniprot23,24 and NeXtProt25) was investigated. Comparative analysis revealed that the percentage of differentially expressed proteins involved in metabolic processes, intracellular transport of various compounds (e.g. proteins, ions, lipids), protein folding, redox reactions and response to stress was higher in CE than in CM and ER/Golgi fraction (Fig. 3); whereas differentially expressed proteins implicated in proteolytic events, regulation of endopeptidase activity, extracellular matrix organization/ remodelling, migration, angiogenesis as well as signal transduction and cell proliferation were more prominent in CM vs. CE and ER/Golgi. In addition, the percentage of differentially abundant proteins associated with mRNA processing and splicing, protein synthesis as well as organization of actin cytoskeleton was increased in ER/Golgi when compared to the other samples (Fig. 3). These findings further indicate the complementarity of the applied enrichment strategies.
Consolidation of the differentially abundant proteins from all experimental approaches (CE, ER/Golgi and CM) resulted in a total of 614 non-redundant changes (Supplementary Dataset S2). Some proteins (n = 19) were predicted by all 3 proteomics strategies to be differentially expressed and at similar trends of expression (up or down) in the T24M vs. T24 cells (Table 2). These included proteins involved in actin binding such as gelsolin and plastin-3, proteases (cytosol aminopeptidase), but also various enzymes [Glucose-6-phosphate 1-dehydrogenase, NAD(P)H dehydrogenase [quinone] 1, phospholipase D3, and others]. An additional 70 proteins belonging to various proteins families [signaling molecules such as Signal transducer and activator of transcription 1-alpha/beta; metabolic enzymes such as Fatty acid synthase, UDP-glucose 6-dehydrogenase, Aldehyde dehydrogenase, and others; proteins involved in cell interactions such as Annexins (ANXA2 and ANXA6) etc.] were found to be de-regulated and at similar trends of expression by two fractionation strategies. Collectively, agreement in trends of expression and statistical significance between the strategies increases the confidence in individual observations (a total of 89 differentially expressed proteins as supported by at least two fractionation strategies may be considered “cross-validated”, hence of higher confidence). In addition, and as shown in Supplementary Dataset S2, some significant changes supported by one fractionation strategy were also suggested by other strategies and at the same trends of expression in the T24M versus T24 cells, nevertheless did not pass the applied thresholds (of at least 1.5 fold change and/or p < 0.05) in the latter. This observation (applying to approximately 40 proteins per strategy) further facilitates prioritization and establishing confidence in individual findings.
Assessment of the validity of proteomic findings by mRNA sequencing analysis
To further assess the validity of the observed proteomic changes, mRNA sequencing data were obtained from the studied cell lines using different biological replicates. Corresponding transcripts for the vast majority of proteins existed. Specifically, corresponding mRNA sequences were found for 1,358 out of 1,359 proteins detected in CE (>99%); 1,061 out of 1,062 proteins in ER/Golgi; and 811 out of 816 proteins detected in CM (>99%).
Among the 253 differentially expressed proteins detected in CE, 98 were also detected with a fold change above 1.5 at the mRNA level. Of the 217 differentially abundant proteins from the ER/Golgi fraction, 85 were also found to be changed at the mRNA levels (fold change >1.5); while among the 256 differentially abundant proteins obtained in CM, 84 were also found to be differentially expressed at the mRNA level. When combined, a total of 210 proteomic changes can be considered as “verified” via agreement with the transcriptomics results (Supplementary Dataset S2). These 210 “verified” findings included various proteins which were defined as differentially expressed in at least two proteomic experiments (Supplementary Dataset S2; proteins marked in red or blue with asterisk) and also proteins which were predicted to be differentially expressed by one only proteomic approach (CE or ER/Golgi or CM; Supplementary Dataset S2; protein marked in green with asterisk), increasing the total number of “verified” findings based on data cross-validation from 89 (cross-validation based on agreement of at least two protein fractionation strategies) to 253 (agreement of at least two –omics strategies; any of protein fractionation approaches and/or transcriptomics, Supplementary Dataset S2). These “verified” features represent a variety of protein families including multiple signalling molecules (e.g. protein kinase C and casein kinase substrate in neurons protein 2, RAS suppressor protein 1, Tyrosine-protein kinase Yes, Interleukin-8, Macrophage colony-stimulating factor 1, Interleukin-6, Vascular endothelial growth factor C and others), proteases (Cathepsin L1, Cytosol aminopeptidase, Carboxypeptidase A4 and others), components of extracellular matrix (such as Fibronectin type III domain-containing protein 3B, Collagen alpha-1(XVIII or XII) chain, Laminin subunit gamma-1 or beta-1, Metalloproteinase inhibitor 3, and others) and also various enzymes (such as NAD(P)H dehydrogenase [quinone]1, Thymidine phosphorylase, Glucose-6-phosphate 1-dehydrogenase, NADH-cytochrome b5 reductase 3 and others).
Assessment of the validity of the multi –omics approach and its potential application
The validity of the differentially expressed proteins reported in individual proteomics experiments (CE, ER/Golgi, CM) as well as in the integrated “verified” dataset (abovementioned 253 proteins) was evaluated in the context of existing literature. Molecular features associated with BCa invasion or metastasis were retrieved using two independent approaches i.e. the BcCluster database20 (n = 627) and the GLAD4U21 tool (n = 671; Supplementary Dataset S3), as described in Methods. Validity was assessed based on the overlap between our experimental data and literature findings (listed in Supplementary Dataset S3). As presented in Table 3, the percentage of overlapping features between the “compiled” (CE, CM, ER/Golgi, i.e. 614 proteins) dataset and literature, was 8.8% (BcCluster) and 11.6% (GLAD4U); whereas for the verified findings (i.e. 253 proteins), the agreement with the literature data was generally higher (overlap range: 13.0–15.8% depending on the comparison (Table 3).
Considering the increased validity of the latter dataset, these 253 proteins were mapped to pathways using the Ingenuity software. The predicted statistically significant de-regulated pathways (p < 0.05, Fisher exact test) were shortlisted, based on their significance level, and the top 15 pathways with the lowest p-value are summarized in Table 4. As a representative example, we present the IL-8 signalling pathway, which notably was the only one found in the top 15 significant pathways predicted based on the literature data and also significant on each individual proteomics dataset. The graphical representation of the IL-8 signalling pathway is shown on Fig. 4 with the differentially expressed features, as detected per individual –omics method, highlighted. As presented, the molecular coverage of the IL-8 pathway increases through the integrative analysis, further reflecting the complementarity of the different approaches. Furthermore, as shown, the vast majority of molecular changes are considered “verified” (Fig. 4 - red frame). Based on this scheme, the chances that the observed “non-verified” changes (e.g. changes supported by one only –omics approach; purple frame) are valid increase, based on their biological relevance. To test this hypothesis the differential expression of the Vasodilator-stimulated phosphoprotein (VASP) was investigated in a set of invasive and non-invasive BCa tissue specimens by western blot. As shown in Supplementary Fig. S3, in line with the ER/Golgi proteomics analysis, a decrease in the level of this protein in invasive versus non-invasive tumors is suggested.
Omics datasets are mine of information, nevertheless only a limited part of it is finally extracted and further investigated mainly due to challenges associated with: a) establishing reliability of findings (typically large numbers of differentially abundant proteins of low statistical power are identified per omics experiment); and b) developing targeted assays for further measurement of individual features, as a means for their verification. Particularly, frequent lack of specific antibodies and the associated costs of performing immuno-based assays result in only a small number of proteomics findings being ultimately confirmed (typically less than 10 per experiment). These verified findings, even though of high value, are not sufficient to comprehensively describe a disease at the molecular level. However, such comprehensive description is required for the successful application of spherical “systems biology” approaches12. In the case of proteomics studies more specifically, comprehensiveness and proteome coverage are dependent on the applied technique, with different subcellular fractions requiring the use of different enrichment strategies for their efficient resolution. The presented approach involving use of different enrichment strategies as well as transcriptomics, addressed the added value of cross-strategy, cross-omics comparisons and respective investigation of consistency in trends of expression, in increasing confidence in individual findings per omics dataset.
We focused on the analysis of BCa metastasis using a cell line model for the specific phenotype. This constitutes a clinically relevant question, as limited therapeutic options are available for patients with BCa metastatic disease, highlighting the need for development of novel therapeutic targets19.
We placed special emphasis on the investigation of the secreted/ extracellular matrix proteome, considered of high relevance in cancer invasion and metastasis11. In parallel to the classical analysis of CM, we also investigated the ER/Golgi fraction, representing the path of proteins on their way to be secreted26. As recently demonstrated and also shown in our analysis, this latter method is of lower enrichment efficiency for bona fide secreted proteins in comparison to the analysis of CM, nevertheless, it can provide new information and highly complementary results to the latter (CM)27. Even though one cannot rule out the possibility that some of the observed differences (or overlaps) between protein identifications from different fractions may reflect sub-optimal enrichment and/or differences in starting protein amounts (e.g. 5 μg for CE, 3.5 μg for ER/Golgi and 3.75 μg for CM analyzed by LC-MS/MS - Methods), the overall specificity of the employed techniques is supported by the Signal IP analysis and respective analysis of protein abundance per fraction (Supplementary Fig. S2). Furthermore, the overall enrichment efficiency in our study is in line with previously published reports28,29.
Several proteomics studies have been published involving characterization of changes underlying BCa invasion either at the total cell30,31,32 or extracellular proteome33,34,35 using various BCa cell line models. A high overlap between the reported identifications in our study and the existing literature was observed, further supporting the validity of the reported findings. Specifically, our shotgun analysis enabled detection of the majority (at least 69%) of proteins identified in previous proteomics studies of total cell proteome from T24M vs. T2431 and T24T vs. T2430 cells. Along the same lines, the majority of proteins previously identified in CM from T24M and/or T24 cells33,35 were also found in our analysis. These multiple existing studies serve as reference points, nevertheless their findings remain disparate and any potential added value from the parallel proteomic analysis of different cell compartments can be assessed with moderate confidence only.
As the first step in this direction, and to establish the relevance of each individual proteomics dataset, we evaluated our main findings in the context of the existing literature. We used a manually curated database of features (genes, transcripts, proteins) associated with BCa invasion/ progression (BcCluster)20. Importantly, BcCluster lists molecules highlighted from studies with sample size of at least 50, suggesting a high validity of the collected features. The second dataset contains the list of the BCa-associated molecules retrieved using the GLAD4U21 tool, without any sample size selection criteria. The two datasets appear to be highly complementary, with an overlap of 179 features, (corresponding to over 25% of features from each literature set), further supporting the assumption that these two approaches provide a good and comprehensive reflection of the current knowledge. It should be noted, that these literature data used as reference in our study include entries reported from different –omics (genomics, proteomics, transcriptomics) as well as non-omics (e.g. immunohistochemistry) studies, apparently collected under different applied methodologies. Investigation of the inter-laboratory variability reflected in these databases would be out of the realm of this study, nevertheless it is expected that this exists. Even though the latter clearly compromises comparability of different studies, on a positive note, it may also be used as a means to increase confidence in individual findings, based on their detection in multiple studies and under different protocols. Along these lines, multiple observed protein changes included in the individual datasets (CE, ER/Golgi, CM) had already been reported in the context of BCa invasion/ progression or metastasis. Independently of the source of literature data, as aforementioned (Results), overlap ranged from approximately 7 to 15% depending on the applied proteomics strategy.
Taking this one step further, an integration of –omics datasets from different molecular levels (proteomics - transcriptomics) was also performed. For almost all proteins identified by LC-MS/MS, we were able to obtain the corresponding mRNA, which strongly supports the reliability of the protein identification process. Even though, in general a moderate correlation between mRNA and protein expression is reported18,36, the regulation trend was well supported by the transcriptomic analysis for many of the differentially expressed proteins (210 out of 614 (34%); notably for 344 transcripts a >1.5-fold change was not reached, whereas only 57 exhibited opposite trend of expression in the T24M vs T24 cells). It should be noted that the presented transcriptomic analysis has some limitations, mostly as a result of the high costs of the next generation sequencing analysis, resulting in a low number of analysed samples (n = 2 per cell line) hampering the application of proper statistical analysis.
Through the application of transcriptomics, which complements but also verifies proteomics findings, an increase in the number (from 89 up to 253) of cross-validated features obtained in the three individual proteomics experiments could be achieved.
The reliability of these latter “cross-validated” proteins was further evaluated in the context of available literature. An improvement in the agreement with existing literature data is observed (as described in Table 3), indicating the applicability and value of such a multi-omics approach to verify large scale proteomics data. Of these 253 features, 33 (13.0% BcCluster)20 or 40 (15.8% Glad4U)21 have been associated with BCa/ BCa invasion or metastasis. This corresponds to approximately a 5% increase in the overlapping features when compared with the respective overlap of all 614 differentially abundant proteins, identified across the three proteomics experiments. This increase appears to be significant, considering that the “verified” dataset consists of a lower number of proteins (253) compared to the combined “all differentially expressed proteins” dataset (614). In other words, the presented strategy facilitates shortlisting more confident findings, which currently range from the small number (regularly less than 10) of verified findings via typical targeted analysis, to the whole list of differentially expressed features per omics experiment (regularly prone to many false positives). The described cross-omics comparison offers the valuable intermediate step between these two extremes, allowing to maximize extraction of features of increased confidence for their further use as input data in systems biology approaches.
As an example in this direction, pathway analysis was conducted. IL-8 signaling was selected, as being predicted (at high significance levels) to be affected based on all, literature mined datasets as well as individual proteomics datasets (CE, ER/Golgi and CM). As presented in Fig. 4, the integrative analysis of –omics data provided a fairly comprehensive molecular phenotype underlying the pleiotropic effects of IL-8 function: The up-regulation of IL-8 in the T24M cells was associated with an up-regulation of matrix metalloproteases (MMP2), implicated in tumor invasion37, as well as VEGFC and ICAM1, factors implicated in angiogenesis38,39 (Fig. 4). Interestingly, the overexpression of MMP2 was accompanied by the down regulation of TIMP metallopeptidase inhibitor 3 (identified in CM analysis), further supporting the activation of MMPs in the context of BCa invasion. Even more: data integration from the different preparation methods (CE, ER/Golgi, CM) links disparate observations revealing events in cases not associated with BCa yet. As shown on Fig. 4, formation of chemosynapse is predicted based on the observed proteomics changes (involvement of VASP, LASP-1), with anticipated impact on focal adhesion and cell migration40,41. In addition, interestingly, involvement of PLD3, a non-classical member (as it lacks lipase activity) of the phospholipase D family of enzymes42 is predicted. PLD enzymes have been implicated as key components of HRAS signaling in cancer cells43 –with, notably, HRAS also detected at different levels in T24M versus T24 cells, based on the proteomics analysis (Fig. 4). In addition, PLD3 has been recently shown to be involved in hypoxia-induced lipid metabolism in colorectal cancer cells44, suggesting collectively, that it merits further investigation in BCa. In parallel to these effects, IL-8 signaling also occurs through G protein coupled receptors, specifically in our system, through Guanine nucleotide-binding protein subunit gamma-12 (GNG12), not studied in BCa yet. Impacts on regulation of calcium channels are expected45,46. Of note, some calcium channels were found at differential levels in the T24M versus T24 cells based on the proteomics analysis e.g. Plasma membrane calcium-transporting ATPase 1 (CE and ER/Golgi), Calcium-binding mitochondrial carrier protein SCaMC-1 (CE)-Supplementary Dataset S2.
Collectively, through the proposed combined analysis of multiple cellular fractions and molecular levels, these multi-level pleiotropic effects of IL-8 previously described in different publications (reviewed by Waugh et al)47 can be better reflected at the molecular level, encompassing changes at the extracellular space (e.g. IL-8 differential abundance), all the way to the nucleus (e.g. changes on Bax; Fig. 4). There is no doubt that multiple missing links still exist nevertheless, such an approach obviously increases coverage (hence confidence), but also facilitates definition of targets for further verification. To better explain this point, the example of the VASP, a protein involved in cytoskeleton remodeling41 and not yet associated with BCa was provided. Being differentially expressed in the ER/Golgi fraction (only), VASP was not included in the shortlisted proteins (i.e. the 253 cross-verified findings). Nevertheless, based on its biological relevance to the IL-8 pathway, the chances that this finding from the ER/Golgi analysis was not a false association increased. Indeed, by using western blot analysis, our preliminary results further supported the down-regulation of VASP in muscle invasive BCa, a finding which we currently further investigate.
In conclusion, our study collectively shows that comparative and in parallel analysis of multiple –omics (in our case: proteins identified in CE, ER/Golgi and CM and also at a different omics level - transcriptomics) has added value on two very important aspects; it can improve proteome coverage and fill missing links, through the complementarity of different techniques. Even more, it can increase validity of individual observations, by cross-omics correlations, facilitating prioritization of findings and ultimately knowledge extraction. Considering the general low statistical power of individual –omics investigations (high number of variables, small sample sizes) such a cross-omics and platform analysis appears a safe way forward particularly towards development of disease molecular models based on valid experimental observations.
T24 and T24M31 BCa cells were employed as described in Makridakis et al31. Briefly, cells were cultivated in DMEM medium (High Glucose, GlutaMAX™, Pyruvate) supplemented with 10% FBS and 1% Penicillin-Streptomycin (P/S) and harvested using 0.05% trypsin/0.02% EDTA and centrifugation (1,000× g, 5 min, room temperature). Cell pellets were washed twice with PBS and stored at −80 °C until further processing. Each experiment was repeated in five replicates (five different flasks with cells originated from same initial stock) per condition.
Collection of secreted proteins from conditioned medium (CM)
CM was collected are described previously27,35 from 10∙106 cells after 24h incubation in serum deprived medium. Protein extraction was performed as described in Latosinska et al27. 75 μg of proteins were processed by Filter Aided Sample Preparation method (FASP), as described below.
Enrichment in Endoplasmic Reticulum/ Golgi Fraction
20∙106 cells were used in order to enrich for ER/Golgi as described by Sarkar el al.26 with minor modifications27. Sequentially, samples were depleted in nuclei (3,000 × g, 10 min) and mitochondria (10,000 × g for 10 min) leading to enrichment for ER/Golgi (16,000 × g for 30 min). Pellet containing the final fraction was dissolved in buffer containing 7M urea, 2M thiourea, 4% CHAPS, 100 mM DTE and 1% ampholytes. 70 μg of proteins were processed by FASP.
Preparation of total cell extract
4∙106 cells were harvested and cell pellet was re-suspended in 200 μL of lysis buffer (7M urea, 2M thiourea, 4% CHAPS, 100 mM DTE, 1% ampholytes). Cells were disrupted by water bath sonication for 10 min followed by centrifugation (16,000× g, 10 min, RT). 100 μg of proteins were processed by FASP.
Filter aided sample preparation (FASP)
FASP was performed according to Wisniewski et al48 with minor modifications49. Briefly, sequential buffer exchange with urea buffer and ammonium bicarbonate (after alkylation with 100 μL of 0.05M IAA) was performed in Amicon Ultra Centrifugal filter devices (0.5 mL, 30 kDa MWCO, Millipore) at 16,000× g for 15 min at room temperature. Proteins were digested overnight on filters with 1:100 trypsin to protein ratio. Centrifugation and lyophilisation were then applied49.
Lyophilized peptides were re-dissolved in 100 μL of HPLC grade water. Subsequently, 5 μL of the peptide mixture was analysed on a nano-flow system (Dionex Ultimate 3000 RSLS, Dionex, Camberley UK), as described before27. Briefly, samples were loaded onto a Dionex nano trap column (C18, 0.1 × 20 mm 5 μm) at a flow rate of 5 μl/min in 98% 0.1% formic acid and 2% acetonitrile, followed by elution onto an Acclaim PepMap nano column (C18, 75 μm × 50 cm, 2 μm 100 Å) at a flow rate of 0.3 μl/min. Reverse-phase chromatography was performed using a linear gradient of solution A [0.1% formic acid and acetonitrile (98:2)] and solution B [0.1% formic acid and acetonitrile (20:80)]. Separation was initiated using 1% solution B (5 min) followed by a gradual increase to 30% (400 min) and 50% (480 min). Ionization involved a nano electrospray source (Proxeon, Thermo Fisher Hemel UK) in a positive ion mode and MS/MS an Orbitrap Velos FTMS (Thermo Finnigan, Bremen, Germany). Ionization voltage was 2.6 kV and the capillary temperature was 200 °C. The mass spectrometer was operated in MS/MS mode scanning from 380–2,000 amu. The MS analysis was performed using a data-dependent acquisition (top 40). Changes between MS1 (MS) and MS2 (MS/MS) modes were done at 60,000 and 7,500 resolution respectively. Parent ions were fragmented at and energy of 40 by higher energy collision-induced dissociation (HCD).
The analysis of the raw MS data files was performed using Proteome Discoverer (PD) v. 126.96.36.1998 (Thermo Scientific). An event detection node was used at a setting of 2 ppm. The Human Swiss-Prot Database24,50 with 20 277 canonical sequences only (downloaded at 30/10/2013) and the Sequest search engine51 were employed. The following criteria were applied: a) precursor mass tolerance 10 ppm, b) fragment mass tolerance 0.05 Da, c) fix modifications: carbamidomethylation of cysteine, d) variable modifications: oxidation of methionine and proline, and e) allowed missed cleavages: one. The false discovery rate (FDR) evaluation was performed by using the Percolator node52 (PD 1.4).
Protein identification and label-free quantification
Protein identification was based on the rank 1 peptides allowing for mass deviation below 5 ppm and FDR below 1%. Only proteins identified with at least 2 unique peptides in individual samples were included for further analysis. The label-free quantification was based on the peak area (i.e. area under the curve), determined based on the extracted ion chromatogram (Precursor Ions Area Detector node in PD). Quantification at the protein level was based on the top three peptides per protein calculated by PD. For the few cases where the protein area was not calculated by the software, as a consequence of lack of integration of the peptide area (a software error), the average area for the particular protein per studied group (T24, T24M) was assigned. In the case of proteins not identified in a particular sample, the missing value was replaced by zero. Twelve proteins derived from the FBS53 or reagents used for MS were excluded from analysis as potential contaminations (Supplementary Table S2). A part per million (ppm)-normalization was conducted as follows: (equation 1), where the total area was defined as a sum of protein areas in each sample. Statistics was performed using SPSS Statistics 17.0 (Mann-Whithney U Test) and R-Package (Benjamini-Hochberg correction).
Total mRNA sequencing
Total RNA from T24 and T24M cells was isolated from 10∙106 cells by TRI Reagent (Sigma Aldrich) (2 replicates per condition) and obtained RNA extracts were purified with RNeasy cleanup kit (Qiagen) including prior digestion with DNase I; both steps were performed according to manufacturer’s protocol. The preparation of libraries and sequencing of the mRNA along with the analysis of the raw data was performed by GenomeScan B.V. The RNA concentration was assessed using the Life Technologies Qubit. Further evaluation of the quality and integrity of isolated RNA was conducted using Agilent Bioanalyzer. Subsequently, samples were processed by Illumina® mRNA-Seq Sample Prep Kit according to Illumina protocol. Briefly, mRNA isolation was performed using oligo-dT magnetic beads followed by mRNA fragmentation and cDNA synthesis. For the latter, the quality and yield was measured via Lab-on-a-Chip analysis (expected product size: 200–500 bp). Clustering and DNA sequencing were performed using Illumina cBot and HiSeq2500 in line with manufacturer’s instructions at the concentration of 16pM of DNA. Image analysis, base calling and the quality check were conducted using the Illumina data analysis pipeline RTAv1.18.64 and Bclfastqv1.8.4. Data obtained from the HiSeq2500 in fastq format was used as source for the downstream data analysis. Alignment of fastq reads was performed using TopHat version 2.0.1254 against the assembled human genome GRCh37.p13 with the corresponding Ensembl release 75 annotation55 (http://grch37.ensembl.org/index.html). The alignment run involved default parameters but allowing for a genome multihit search and transcriptome build and mapping. Alignment quality metrics were collected using Qualimap version 2.0.156. Quantification of feature alignments was performed using HTSeq-counts from HTSeq framework version 0.6.1p157. Default parameters were used for a non stranded RNA-seq library using the intersection non empty algorithm. Normalization of the count data and statistical analysis for the differential expression was performed with DESeq2 package version 1.6.358 for R statistical computing software59.
BCa tissue specimens were collected in Germany (Department of Urology and Urological Oncology, Hannover Medicine School) from patients undergoing resection of the bladder. All individuals gave written informed consent. All experimental protocols for tissue sample collection were approved by the Hannover Medical School Ethics committee (case number: 614–2009) and experiments were performed according to relevant guidelines. Specimens from non-muscle invasive (n = 3), muscle invasive (n = 3) BCa and negative biopsies (n = 3) were analyzed. Tissue lysis was performed as described earlier49. 20 μg of total protein per extract were separated by NuPAGE® Gradient Gel 4–12% under reducing conditions and electroblotted to nitrocellulose membrane (LG), as presented elsewhere60. Membranes were incubated overnight at 4 °C with the primary mouse anti-VASP antibody (Enzo LifeScience, ALX-804-177-C050, dilution 1:500) or anti- β-actin antibody conjugated to HRP (Santa Cruz, sc-47778 HRP, 1:4,000), in the first case followed by incubation with anti-mouse HRP-conjugated secondary antibody (Santa Cruz; dilution 1:2,000) for 2h at room temperature. Target protein was detected by Enhanced Chemiluminescence (Perkin-Elmer LAS, Inc.).
Molecules (proteins and transcripts) associated with BCa invasion/ progression were retrieved from the BCa database (http://bccluster.org/)20. GLAD4U21 was also employed to retrieve relevant featured from MEDLINE database using the following keywords: (“bladder cancer” or “urothelial cancer” or “transitional cell carcinoma” or “urothelial cancer”) and (“invasion” or “progression” or “invasiveness” or “aggressiveness” or “metastasis”) with the undefined threshold settings for genes prioritization.
The biological function of the differentially expressed proteins was manually evaluated based on the Gene Ontology (GO) annotations retrieved from Uniprot-GOA annotations23 and/ or NeXtProt database25. In parallel, differentially expressed proteins which were considered as “verified” were mapped to pathways using QIAGEN’s Ingenuity® Pathway Analysis (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity). Statistical analysis was conducted by using right-tailed Fisher’s exact test. Pathways with a p-value below 0.05 were considered as significant.
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The work was supported by the BCMolMed grant (PITN-GA-2012-317450) from the FP7–PEOPLE–2012–ITN program and in part by the TransBioBC FP7 Health project funded by the EU Commission (grant agreement 6019330).
Harald Mischak is the founder and co-owner of Mosaiques Diagnostics. Maria Frantzi is employed by Mosaiques Diagnostics as part of an EID (Industrial-Academia) Marie Curie Action. Bart Janssen and Daniel M. Borràs are employed by GenomeScan B.V.
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Latosinska, A., Makridakis, M., Frantzi, M. et al. Integrative analysis of extracellular and intracellular bladder cancer cell line proteome with transcriptome: improving coverage and validity of –omics findings. Sci Rep 6, 25619 (2016). https://doi.org/10.1038/srep25619

domain: biology


text:
Voting has commenced in the Off Cycle Governorship Elections in Kogi, Bayelsa and Imo States.
Election materials and Ad-Hoc staff of the Independent National Electoral Commission arrived the various polling units across the Three States early with voters getting accredited and voting simultaneously.
In Bayelsa State, Governor Duoye Diri who is contesting for a Second term in office voted at about 8,45am in the morning.
He commended the process describing it as seamless while also commending the Independent National Electoral Commission for the seeming improvement in the process.
He also raised his concerns about voter intimidation in some parts of the State calling on Security agencies to ensure that purveyors of violence are dealt with decisively.
There has been no report of incidents yet as of the time of this report in any of the States with accreditation and Voting going on simultaneously across polling units.


text:
The short-video platform’s stated goal of promoting “a civilized lifestyle” aligns with an ongoing government campaign to make China’s cyberspace more positive and wholesome.
If you live in China, flexing with a fat wad of cash can get you banned on TikTok.
In a notice Friday, Douyin — as the enormously popular short-video app is branded in China — said six kinds of content including “flaunting wealth” are now banned on the platform because they promote “unhealthy values.”
The content prohibitions follow the introduction of livestreaming regulations that highlighted wealth-flaunting as an issue last year, as well as the government’s recent call to spread more “positive energy” online.
The six similar categories identified by Douyin also include promoting money-worship, using minors in videos related to luxury products, and “showing off one’s social status in an inappropriate way,” which includes “making fun of the poor.”
Fabricating narratives — ascending from rags to riches or having just returned from studying abroad, for example — in order to market products or scam users makes up another banned category.
To help get the message across, the notice includes descriptions of disallowed content that resulted in account deletions, such as depictions of large amounts of cash being thrown into the air.
The purported aim of the ban is to promote “rational spending” and “a civilized lifestyle,” as well as build a healthier community on Douyin, according to the notice. A spokesperson for the platform told media that flaunting wealth “pollutes the social atmosphere (on Douyin) and is particularly harmful for the mental and physical well-being of minors.” Continue to read the full article here
– This article originally appeared on Sixth Tone.


text:
Houzz is the new way to design your home.
695,371 landscaping house Home Design Photos
Tuckahoe Creek Construction, Inc.
Marcia Fryer did design the landscape, the hardscape and the pool.
it.” A new standing-seam copper roof extends over the new patio and part of the house. The chimney marks where the house ends and the new covered patio begins.While the new structure is more rustic than the rest of the house, it still ties into the architecture. Compare the detailing at the peaks in the
the previous photo with this one to see how the new timbers emulate the original design.Landscape architecture by Marcia Fryer
VanBrouck & Associates, Inc.
French Country Estate
what is the stone on this house
like the front door section of this house on the Nims mansion
like the shape of house
Build this house...Exterior House
Smith & Vansant Architects PC
Shingle style home in Hanover NH
Any ideas of the paint color? Love this house!
this house is so amazingly beautiful! I am in love. It would be much to much house but if I could scale it down it would be perfect. You guys are amazing!
LOVE the colors. What kind of stone is that on the landscaping walls. Very pretty. Is this real cedar on the siding and slate on the roof? Thanks!
love red window trim, house colors and window mullions in only upper part of window
i like the tiered look of the back of the house as it goes down the hillside, and the colors/materials of the exterior. and the dormer/gable on second floor
I have an early 1950's L-shaped ranch house built into a hill. Looks like a one story from the front, but from the garage side and back of house, it looks like a two story. I really like the look of B&B siding, but can't decide if it would look
siding of house, tin roof with french doors open
Can we do this to our house!
This reminds me of my Grandparents house at Manawhai.
what about all across back of house?
the guesthouse and to design a structure that would match the character of the main house," Dussouchaud says. The main house (not shown) is a traditional California-ranch-style home, with large patios around the house and white board and batten siding. Both buildings have roofs made of composite shingles
Vinci | Hamp Architects
Where is this house located? What style of house is it?
Thanks for your question. The stone tiles measure approximately 23" x 45. Inside the house, the flooring is terrazzo. Good luck with your project.
Patio - I like this because it kind of matches the rest of the house, and it has the same landscaping.
This view within the courtyard shows how open the house is toward it. The ground floor has sliding glass walls connecting inside and outside, the stair core has plenty of glass facing south, and a series of terraces on the second and third floors face the courtyard as well.
Simpson Design Group Architects
Where can I get that front door & the house numbers?
Chad, That would have been decided by the landscaper, and we do not have that information on file here. Sorry we could not be of more assistance.
Thanks houseArt, for the recommendation! We do have a door very similar to the one above. It's called the
Pinnacle Mountain Homes
411 Long Ridge
Give me project of this house , please
I really like this house, I won't to build the same
House stamped concrete
Outside of house cabin
Modern/ traditional house
mountain house...mountain landscape...rock landscape...Back of house...Dream house
Raymond Jungles, Inc.
Inspiration for small modern pavilion project. Having the outside around the house but then having an extra layer, making the whole property more private...yet it is still outside. The water element, making the steps float creating a walk to the front of the house.
Controlling the access to your house can be an important security benefit that the modern moat can provide, even when the moat is within the house's perimeter, as shown here.These randomly shaped stepping stones lead over the moat, fulfilling its traditional purpose of blocking the entrance but with
Controlling the access to your house can be an important security benefit that the modern moat can provide, even when the moat is within the house's perimeter, as shown here.These randomly shaped stepping stones lead over the moat, fulfilling