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Stumped? Ask away
A forum for discussing travel both to Southeast Asia and beyond. Good, useful information for those planning (or returning from) a trip to anywhere in Asia and Australasia.
Use the quicklinks below to jump to a specific part of the forum below. All sections can be read by anyone, but you need to be logged in to be able to post a question (or answer). Not a member? You can become a member here.
Brunei - Trip Planning
By floriankappel on 12 Oct 2016
Should tourists still visit Burma?
By DLuek on 22 Nov 2017
Boat tour north of Luang Prabang on the Mekong River
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Bali itinerary - 16 days between different places
By somtam2000 on 24 Nov 2017
No more boat travel between Nong Kiaow and Muang Khua
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Melaka Update
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Cebu city to malapascua
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5 days in Singapore
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Early Itinerary Planning for SEA
By amnicoll on 13 Nov 2017
Anyone ever been here?
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From Phuket airport to Koh Jum
By DLuek on 24 Nov 2017
Mekong River Cruise from Phnom Penh to Saigon
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Border crossings Thailand to Burma E-Visa
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Grand adventure in Gran Canaria
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The Vietnam bucket list
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1 month stay in china.. help
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Is there any Hong Kong Tourism Guide ?
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JR pass: 7 or 14 days? Itinerary set
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Information source for independent travel/living/working in Mongolia
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If I were gonna do a tour in North Korea...
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Roaming wifi in Korea
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3 months in Taiwan - I'm here already
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Travel companions needed - British passport holders
By JLam on 26 Jul 2014
SEA 2017 July/August help
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By myjimmy on 28 Jun 2017
Super malaria rises in Southeast Asia
By somtam2000 on 22 Sep 2017
Update on Travel Insurance
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Food and drink budget
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Can you use a Verizon iPhone on GSM ?
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Photo sharing sites
By braylor on 22 Nov 2016
3 weeks in Cambodia/Laos with 7 year old- does our plan look OK?
By FoundVagabond on 12 Aug 2017
Dressing as a tourist.
By myjimmy on 27 Jun 2017
Authentic travel
By somtam2000 on 7 Oct 2017
One Month Cycling through SE Asia: Best Route Advice!
By DonRuperta on 27 May 2017
HCMC to Hanoi.
By Landylordy on 15 Jan 2017
Diving/snorkeling with sharks in August
By Aehui777 on 23 Apr 2017
volunteering in Vietnam
By idex on 26 Apr 2016
Telling the Time in Thailand
By daniel969 on 23 Jul 2015
Trekking in Mondulkiri
By AnnMarieHall on 6 Sep 2017
La Balian the new place in Balian can I list it with you please?
By LaBalian2015 on 27 Jun 2017
Pakse backpacker accommodation
By muzzaf on 24 Oct 2017
Dorms KL 2 Backpacker Hostel
By dormskl2 on 11 Apr 2017
Biking Singapore Cycling Tours
By ulas1907 on 7 Nov 2015
Maraya Hotel & Resort - romantic riverside resort in the city of Chiang Mai
By fotofancy on 11 Sep 2017
Hanoi Gratitude Hotel - brand new Hotel in hanoi Old Quarter
By hanoigratitudehotel106 on 23 Aug 2017
Unexpected downtime
By somtam2000 on 27 Oct 2017


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Welcome to Crazy Baby Clothing, where things can get a little crazy, as they so often do with a baby!
Explore our wide range of hip and spunky baby and toddler products, or travel in style with one of our heavyweight canvas diaper bags or other luggage items.
Featured Collections
Punisher Collection
Now even your little one can proudly wear ever popular Punisher gear.All Punisher Collection

subset: commoncrawl


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Joined: 26 August 2006
Posts: 2690
hi guys
this question is 4 mosta people hu watched India-Calling!
u knw, guyz lke Adi have a way ov sayin' stuff lke da way he says,"Adi rocks!". when hez leavin', lke sayin' Bye..how duz he say it??..what is da word he uses??
[[i knw sum ov u people mite b thinkin wht a stupid !but seriouslii,yesterday wen i woz lke leavin' nd i sed "adieu" 2 mah sis nd c goes..hahaha u copied Adi nd m lke wht da hell??..i neva..he says adi rokz!..nd i asked her if c even knws wht adieu mean then c says it means bye nd Adi says it;u copied him!but i dint so we'd a bet so dunt wanna lose!plz tell meh]]
thnx annie~
Joined: 26 August 2006
Posts: 2690
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Los Angeles, Sep 25 (IANS) Juan Manuel Gomez Fernandez and Juan Manuel Gomez Monteverde, the father and brother of American actress Ali Landry's husband Alejandro Gomez Monteverde, were found dead over the weekend in Veracruz, Mexico, after being held hostage for 16 days.
The two were reportedly kidnapped outside their house in Tamaulipas on September 4. They were later discovered with severe head wounds in the remote town of Pueblo Viejo.
According to Televisa media company, the kidnappers did not release them although a ransom had been paid, reports acehshowbiz.com.
Landry, a former Miss USA who has starred in films like "Who's Your Daddy?", "Bella" and "Crescendo I", and her filmmaker husband spread the ashes of the deceased off the coast of Tampico, Mexico, on Wednesday.
A mass in honour of the two men will be held in a church here on Friday.
The family hasn't issued a statement about their loss, but Eduardo Verastegui, the producer who worked with Monteverde on the film "Little Boy", expressed his heartfelt condolences on Twitter.
"With a heartful of pain and sadness, I ask for prayers for my friend and 'compadre' Alejandro Gomez Monteverde and all his family," his tweet read in English.
"My deepest condolences to my soul brother, I love you so much 'compadre,' I put my heart in your hands and I join you in this deep pain," he added.


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Artist description:
I did some food girls while one of my professors was lecturing. I translated one of the sketches into this and I might do the same to the others, not sure yet... I might do Suhi girl next or maybe miss Cotton Candy. :)
Java Draw Created Sep 25, 2012 460 x 640px Unless noted Copyright 2012 tomorrowNight.
We encourage you to submit a comment and let the artist know what you think of this drawing. Please log in or join to post a comment.
Post a comment
This is great,, so fun and colorful,,, great work
sooo glad u did cotton candy next hehe :p n wats suhi?? da colors r sooo pretty here tho n i luv her outfit design--favers :3
Super drawing!:)5h
I scream for ice-cream! Delicious colors
so sweet ! love the colouring! 5♥s
the color choice is excellent and the details too,u are having such cool ideas:) keep it up 5
I LOVE the colours and details in this series! You are sooo talented! 5/5
so creative and cool! she's so sweet and what a great coloring!
omggggggg awesomeeeeeeeeeeeee.5/5.


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Netflix Shares Underperforming Today, Could Be Related To Concerning Comments From Bearish Bernstein Analyst
Traders have been fading the morning rally in shares of Netflix (NASDAQ: NFLX) Monday. Some traders in the name are attributing the weakness to some comments from Bernstein's Carlos Kirjner, a bear in the stock as of June 25th.
After opening at $310, stock has now lost all of the gains and is trading around $305.15, down around $0.50 for the session. The NASDAQ is trading up more than 13 points, or around a third of a percent.
In the note, Kirjner said, "We believe Netflix may reach 40-45 million domestic subscribers in the fullness of time as its addressable market is limited by broadband penetration and quality and by the availability of Internet connected TVs or "bridge devices" such as game consoles." The analyst had a similar sentiment on Netflix's international opportunities.
Kirjner highlighted, "the current valuation implies a domestic subscriber base well above our 40-45M estimate and/or a higher monthly fee under the assumption that the service is or will be largely price-inelastic."
Bernstein's Kirjner maintains an Underperform rating and $180 price target. The analyst's price target represents potential downside of more than 40 percent from where Netflix shares are currently trading.
BENZINGA NOTE: Kirjner has never been a bull on shares of Netflix. The analyst initiated coverage on Netflix at the beginning of November in 2011 with a Market Perform rating.
Latest Ratings for NFLX
|Oct 2017
|Loop Capital
|Oct 2017
|Oct 2017
View More Analyst Ratings for NFLX
View the Latest Analyst Ratings
Posted-In: Analyst Color News Analyst Ratings
© 2017 Benzinga.com. Benzinga does not provide investment advice. All rights reserved.


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Accessory Concierge - Jaw-Dropping Jewelry at a Price You Can Afford8:34 am
Whether you're rocking a BoHo chic, avant-garde or romantic look, all your jewelry dreams come true with Accessory Concierge.
To keep my look fresh, I shopped Accessory Concierge for jewelry that expresses my style and updates my wardrobe (hint: you can wear the same black dress for ten years if you simply change up your jewelry).
My style icons - Jessica Alba, Joan Smalls, Charlize Theron, Lupita Nyong'o, Emma Stone,
Liya Kebede, Michelle Obama, Kate Middleton - all rock a look that follows them from outfit to venue, stage to screen. And they share similar taste - modern, chic and very streamlined, with a slight edge.
It's what I try to emulate in my own very non-famous life.
For big events, you'll almost always find me in a shift dress with a statement necklace. It's how I feel rich without being over the top (and it's easy to get ready in a flash). Accessory Concierge's Arctic Brass Collar (pictured above) is so jaw-dropping, I've wore it to every event I had for the last three weeks. The mix of crystal and brass has a simultaneous look of both the 40s and so-right-now. And it looks as expensive as J. Crew and Banana Republic's ones that run up to $200, but it less than a quarter of the price! Truthfully, I love it so much, I would sleep in it if I could.
I always dress for the occasion and crowd, so if I want something with a bit of edge, I rock my Rawcut Amethyst Druzy Pendant. The colour and natural ombre of the amethyst is stunning and I am smitten with the unique chain that is simultaneously delicate and very 1950s. And no matter what colour I've worn it with, it looks s-p-e-c-t-a-c-u-l-a-r.
Having short hair and thick rimmed glasses, I like to keep my earrings pretty simple, and almost always studs. But of course I want them to be stunners, nonetheless. Here are the beauties I am seriously coveting right now:
- Sadie Studs - So much more elevated than my usual plain gold studs, they go with everything and look so luxe despite the affordable pricetag (p.s. I wish they came as a lamp base too).
Having short hair and thick rimmed glasses, I like to keep my earrings pretty simple, and almost always studs. But of course I want them to be stunners, nonetheless. Here are the beauties I am seriously coveting right now:
- Sadie Studs - So much more elevated than my usual plain gold studs, they go with everything and look so luxe despite the affordable pricetag (p.s. I wish they came as a lamp base too).
- Pave Chevron - I can't stop wearing these. They are so freaking beautiful. The construction is perfect - no missing or half stones, no dark background peeking through like the ones I've had in the past. They are on trend, but remain sophisticated, so they work with everything from jeans and a tee to a LBD. And they're available in both gold and silver. Swoon.
- Fulham Studs - I am a huge fan of the bar earring trend. The minimalist line design fits right in with my preference for looking uncluttered. These take the look up a notch with precisely placed crystals and half inch long 14k gold plated bars, complete with sleek geometric finials. I customize them by changing their position - horizontal, straight up or on a slight slant, following the edge of my ear. Amazing go-tos for life.
- Pave Bar Studs - Extending the bar even more, these elegant studs stretch just long enough to grab your attention, without being dangly or distracting. In other words, they're perfect. They upgrade whatever I'm wearing and are suitable for meeting friends for a casual brunch to a fancy press dinner with dignitaries.
- Tambora Pyrite Studs - While the metallic polish of these hunky rougly-hewn cubes might not be actual gold, they sure look like it (the posts are, with 24k gold fill). The shimmering squares look like they weigh a ton, but are actually super light - phew. Luminescent and glorious, they are simply magical.
Accessory Concierge is one of my best finds all year, so shop now and get absolutely radiant jewelry that makes you feel like a billion, without dropping even a hundred.


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Say it ain't so!
After 30 years together, one of Hollywood's most loveable couples is officially calling it quits.
Danny DeVito and Rhea Perlman have officially confirmed that they are separating, according to a rep for the pair.
Danny, 67, and Rhea, 64, married in 1982 and share three children together: Daniel, 25, Grace, 27, and Lucy, 29.
The couple met in 1970 when Rhea saw Danny in the off-Broadway play, The Shrinking Bride. Years later, Rhea landed a part as Danny's girlfriend on the hit show Taxi. They have been living together for 11 years.
Though they are definitely breaking up, we'll "cheers" to Danny and Rhea's unforgettable iconic on-screen chemistry!


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Available from No Starch Press (print copy comes with free ebook) or from Amazon.co.uk, Amazon.com, Barnes & Noble and many more booksellers.
All of this book's featured examples are available on Github, and you can download all the examples as a single Zip file. An updated copy of Appendix A is available, and more companion assets are coming soon.
"Peter Gasston has now done for the modern web platform what he already did for CSS: write a consult-it-first compendium of information for web developers of practically any skill level."— Stephen Hay, author of 'Responsive Design Workflow'
Learn how to:


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Scientists at NUI Galway may be able to reduce the rejection rate of corneal transplant operations, meaning the number of people rendered blind from corneal eye disease may decrease.
The research, which uses stem cells, could lead to more patients holding onto their sight, if the technique can be replicated in humans.
Corneal eye disease is the fourth most common cause of blindness, affecting ten million people worldwide.
Corneal transplants are the most common form of treatment for the condition, and involve the replacement of the damaged cornea with healthy tissue from an organ donor.
Although 100,000 people undergo corneal transplants each year, the procedure suffers from a high rejection rate of 30%.
However, researchers at the REMEDI regenerative medicine institute at NUI Galway have developed a new system which could reduce rejection rates to as low as 10%.
The lab based study, details of which are published in the American Journal of Transplantation, involves the use of a type of stem cell grown from the bone marrow of adult donors, which when administered adjusts the immune system's balance, reducing rejection.
A follow up €6m EU funded study is now under way to examine the issue more closely, and will involve testing the research on humans in clinical trials.


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New structure for European aviation regulators?Commissioner Barrot seeks views of European governments and industry
(20.09.2006) -- In an unprecedented initiative, Commissioner Barrot convened leaders of Europe's aviation industry together with their regulators in a one-day High-Level Conference in Brussels today, 20 September, on the theme of 'The Future of Aviation Regulation in Europe'.
Related Research on ASDReports.com:
The Global UAV Market 2015-2025
The Global UAV Market 2015-2025
Representing Europe's major network airlines, Ulrich Schulte-Strathaus, Secretary General of the Association of European Airlines, presented his vision of a regulatory landscape tailored to the needs of a key European industry and its customers.
"Our airlines want to offer their passengers a safe, direct, comfortable and affordable flight from A to B", he said, "and yet their costs are increasing because of an array of national, pan-European, EU and international agencies that all regulate aviation in Europe and levy all sorts of fees and charges. We have one Single European market for 25 European sovereign states."
The AEA has commended the Transport Commissioner for "being serious" about a dialogue between the European aviation sector and the EU Commission on how Europe's competitiveness could be enhanced. "The consultation with European Member States and industry is a "methode a la Barrot" which should become institutionalised throughout Europe," said Schulte-Strathaus. "We have regulators at regional, national and EU level. But we actually need only one European safety agency, like the USA, for one single (European) market. And we have industry involvement in the creation of one Single Air Traffic Management in Europe for one Single European Sky. Surely there is room for simplification."
The AEA feels that regulators should "facilitate" the task of airlines to provide safe air services in open and fair competition for European consumers. "That is the added value we expect from the European Union for Europe's aviation sector - to help us become sustainably competitive globally".
Source : Association of European Airlines


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Arabia Azur Resort Hurghada
El Cornich Road, Hurghada, Egypt
The elegant Arabia Azur Resort offers 300 bright guest rooms as well as a cash machine, a designated smoking area and a safe deposit box.
The property lies within 2.1 km from Giftun Island, Eastern (Arabian) Desert and New Marina. The venue is set within 4 km from Hurghada city center.
Set close to coral reefs and gardens, this hotel offers access to a beautiful beach with beach chairs, a beach bar and towels.
Arabia Azur Resort features rooms with a contemporary interior that come with a dressing area, a private safe, a living area, a private balcony and TV with satellite channels for a pleasant stay in Hurghada. The property offers views of the Red Sea from the rooms. They are fitted with marble bathrooms with a Jacuzzi, a shower and a hairdryer.
Guests can also enjoy international meals at the delightful restaurant. The sports bar serves snacks and ice cream.
Hurghada airport can be reached in 10 minutes by car from Arabia Azur Resort.
Car rental services are available on site.
Arabia Azur Resort pampers guests with a library and a tennis court. The hotel provides deep-sea fishing, diving and beach volleyball for an active pastime.
Important information
- Children and extra beds
- One child under the age of 5 may stay free of charge when using existing bedding.
- One child may stay at the price of 25 % of the room stay per person per night in an extra bed.
- Maximum capacity of extra beds in a room is 1.
- On-Site Parking
- Paid Wi-Fi
- Express Check-In/Check-Out
- 24-Hour Front Desk
- Family Rooms
- Designated Smoking Area
- Safety Deposit Box
- Business Center
- News Stand
- Terrace
- Garden
- Garden furniture
- ATM/Cash Machine
- Luggage Storage
- Barber/Beauty Shop
- Library
- Multilingual Staff
- Wheelchair Access
- No Pets Allowed
- Breakfast
- Buffet
- Welcome drink
- A la carte restaurant
- Snack Bar
- Children's buffet
Leisure & Sports
- Outdoor Swimming Pool
- Children's Swimming Pool
- Spa & Wellness Centre
- Tennis Court
- Kids Club
- Fitness center
- Water Sports
- Private beach
- Treatment room
- Steam room
- Children's Playground
- Jacuzzi
- Sauna
- BBQ Facilities
- Entertainment
- Turkish bath
- Karaoke
- Beach umbrellas
- Beach chairs
- Towels
- Sun terrace
- Full body massage
- Foot massage
- Back massage
- Head massage
- Beauty treatment
- Waxing
- Manicure
- Facial
- Pedicure
- Body wraps
- Body scrub
- Diving
- Table Tennis
- Windsurfing
- Snorkelling
- Bowling
- Darts
- Billiards
- Board games
- Paid Airport Shuttle
- Limousine Service
- VIP Room Facilities
- Tours/ticket assistance
- Meeting/Banquet Facilities
- Fax/Photocopying
- Newspaper service
- Currency Exchange
- Hairdresser
- Shops/Commercial Services
- Car Rental
- Laundry
- Ironing Service
- Baby-sitting/Child Services
Room Amenities
- Air Conditioning
- Dressing area
- Sitting Area
- Balcony
- Carpeted floor
- In-room safe
- Dining table
- Mini-Bar
- Tea & Coffee Facilities
- Bottled water
- Telephone
- Hair dryer
- AM/FM Alarm Clock
- Beachfront view
El Cornich Road, Hurghada, Egypt
Arabia Azur Resort features rooms with a contemporary interior that come with a dressing area, a private safe, a living area, a private balcony and TV with satellite channels for a pleasant stay in Hurghada. The property offers views of the Red Sea from the rooms. They are fitted with marble bathrooms with a Jacuzzi, a shower and a hairdryer.
Please, select dates to see available rooms.
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Contact Us
Arabia Azur Resort
El Cornich Road, Hurghada, Egypt
What's nearby
- Local attractions
- New Marina1000 m
- Diving Ocean Makadi Bay1000 m
- Shaab Abu Ramada/The Aquarium1.7 km
- Dolphin Dancers2.7 km
- Eastern2.8 km
- Careless Reef2.8 km
- Omo Gamaar2.8 km
- City Center3.7 km
- Airports
- Hurghada7.6 km


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[Verse 1: KYLE]
Alright well she thinks I'm in love right
I shouldn't of gave her that hug, right?
When it comes down to it
Every girl in the world wants me though
Nigga I can look good in a speedo
No lie I can see it in her eyes
She wants D.i.K
From K.i.D doe
Way on top of my A game
Everyone else way lame
Your style has been gone
You arrived on that late train
You think your cool
Like fake chains
Niggas ain't foreally doe
And ain't nobody fuckin' with you
Fake brands of cereal
And I know
What these girls think
They can just fuck with my brain
And Make me do what they say
But they can't
I know
Cuz I'm way on top of my flow
And shes has nuthing that's inside
So I'm out the window
I don't do really girls
Who do nuthin but gossip
Try to say some nice things
Your breath stinks
When you talk shit
And thats real
[Verse 2: KYLE]
Alright well it's K dot i dot D dot damn
Niggas know that I'm so for real
I always kill
And I'm always trill
And I'm always something these niggas feel
I am way up over your head still
I'm stacked up with bread still
Youre headed nowhere
Like running on a treadmill
Niggas FxL is my team yo
Nuthin comes In between bro
Super Mitch and Super Chris
Theyre all addicted to weed though
I am addicted to Gettin it by any and all means
I'm not addicted to girls
But they're addicted to me
Maybe because I got sumthing inside
K.i.D is a hell of a guy
Youre hell of irrelevant
Dealing with I
I'm Super Duper
Nigga right
..... Right?
Oh yeah
Skinny niggas runnin' shit
Im the fuckin' best that's ever fuckin' lived in life haha
Yes, it was directed by Jakob Owens and produced by Nolan Smith.


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" Brand: Asda / Type: Foundation "
I bought this foundation one day when I had forgotten my make-up bag! The foundations were very cheap-looking hanging up on the shelf and not in a proper make-up counter. It only cost £2 for this foundation, and Asda also sell bottle-type foundations for a little bit extra.
The makeup comes in a very, very squishy tube. The reason being is that the foundation is very watery and there's not much for a squeeze needed to get it out onto your hand. Also, you need to put the finger on your face very fast afterwards so it doesn't roll off and away. Perhaps a sponge would be more useful for this!
Do not try to apply too much of this foundation on at the one time as it is very runny and you need to allow it time to dry. The biggest problem I had with this foundation is that the colour was pretty strange. Instead of being a skin colour, or even orange, it was very yellow. Once dried, it looked a little golden which was okay, but it was a very yellow foundation.
Because this foundation is so runny, it is hard to apply this foundation evenly! The coverage was very good, it hide my spots just fine. However, it was a horrible texture on your skin once dry. It made my skin feel very rubbery, and you could see the lines where I had rubbed it.
This was a great option for a one-time stand-in foundation, but won't do for an everyday foundation!
This is ideal for the days/ people who like to wear makeup but you arent going anywhere so there is not a point in wasting expensive makeup. Its cheap at only £1.87 per tube and is ok in quality. It is quite runny so you have to be quick whilst applying it but using a sponge/makeup wedge would help no end. There is only about two different shades of colour and no testers available but at £1.87 you could buy both shades too see which one is for you and you would not really break the bank.
Obviously with makeup you tend to find you have to spend quite a bit for a high quality product and the quailty in't really high with this product but as i said before it's great for days when you're just in the house etc. but still like to wear makeup, that way you'll save your expensive foundation for special occasions.
I was going through the Asda deals last week and saw the 3 for 2's on their new smart price make-up range. Well, as a mum of 5 I thought it might be nice if I can get some make up thats cheap, affordable and doesnt smell like something had died and smeared it over my face for decoration!
Well, I can report that it doesn't smell like a dead carcass, infact it has no perfume. I guess it smells slightly 'talcy' but that's about it. Coverage is smooth, and it dries rather nicely and smoothly, however do use the sponge wedgie applicators as this foundation seems too watery for the dot & smooth technique my mother uses. I lasted roughly 4 -5 hours before starting to go patchy, but as this it usually when I get time to reapply anyway, its no biggy for me. It might be for a working lady/mum, but for the price it does enough of a job for me.
At a rather cheap £1.87 for 20g this foundation comes in translucent & unnamed shades (only 2 shades which is disappointing, but understandable at that price) There's no way to 'test' the colour so your in for pot luck. With my age and the way I caught the sun this summer I can use the translucent and just look slightly tanned (I'm very white translucent skinned normally)
Would be OK for stage too if you cant get hold of the proper stuff.
Oh and for mums.... I have found a little neat washing up liquid or a baby-wipe clears this up quite quick if spilled ;)


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Microsoft confirmed that data on its European servers can be handed over to American investigators because of the Patriot Act. This can be done without notifying European customers. The EU has what's called a Data Protection Directive, requiring customers to be notified but Microsoft says that the Patriot Act trumps any European law.
This is interesting to us over here at Marketplace Tech Report because our story airing today is all about Microsoft going along with Chinese law in censoring search results on Bing. Microsoft says it respects the laws of the countries where it does business and follows them accordingly.
Seems like an inconsistently applied standard.


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WR Schultenover dismissed from Bison football team
FARGO -- North Dakota State wide receiver Jordan Schultenover, who was expected to be the team's top pass-catching target next fall, was dismissed from the team for a violation of team rules.
Bison head coach Craig Bohl would not specify why, saying, "I'm not in a position to make further comment."
Also, Bohl said backup running back Pete Blincoe, who started two games as a sophomore but missed last season because of a knee injury, left the team.
"I think it was one of those things that just wasn't there," Bohl said. "He just felt like it was his best interest to move on."
Schultenover was first in line to replace Kole Heckendorf as NDSU's No. 1 receiver. He played in 32 of 33 games in his career finishing with 19 receptions and six touchdowns.
Schultenover was arrested for minor in possession in 2005 according to Fargo police records, an offense that went previously unreported most likely because he was a redshirt freshman and didn't play at the time.
His situation further clouds a spring that has seen a backup quarterback get kicked off for reasons other than a marijuana possession charge and three players being suspended indefinitely for driving under the influence charges.
Schultenover was an honorable mention all-state selection in Minnesota at Hopkins High School in the Twin Cities. At 6 feet, 2 inches and about 210 pounds with a 4.39-second 40-yard dash, he showed promise during spring football.
Bohl said the program is addressing the recent incidents with education, suspensions and dismissals. Schultenover was not arrested by local law enforcement in connection with any criminal activities as of Tuesday.
"Character is the bedrock of our program," Bohl said. "In recruiting, we ask the tough questions on the front side. We've turned down several players didn't fit in. I'm concerned about the recent number of incidents; it's not consistent with the last six years and 600 players."
Schultenover's departure leaves sophomores Warren Holloway and Sidney Haynes as the starters. Junior Gary Williams is out with an injury.
Bohl said junior college transfer receiver Sam Rogers from West Los Angeles Community College will have a chance of contributing right away.
"We feel like we have some depth coming in," Bohl said. "I just don't think we're going to go run and sign somebody."
Blincoe was attempting to challenge sophomores Matt Voigtlander and D.J. McNorton and freshman Josh Horner for the backup spot to starter Pat Paschall. He had 417 yards rushing on 76 attempts and five touchdowns in 2007.
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A cDNA microarray resource enhanced for transcripts specific to the bovine mammary gland (BMAM) has been developed and used in pilot studies to examine gene expression profiles in the mammary gland. One goal driving development of this resource was to shed some light on the pathways and mechanisms specifically related to bovine mammary gland growth and development. To accomplish this, gene expression patterns from bovine adipose, liver, adrenal, lymph, spleen, thymus, gut, and developing mammary tissue were compared using the BMAM microarray. We have thus identified a putative set of 16 genes being preferentially expressed in developing mammary gland. Another of our long-term goals is to elucidate the genes and pathways associated with bovine lactation and involution and to use these as a model for human mammary gland development as it relates to human breast cancer risks. To begin this process, we conducted a pilot study, comparing gene expression profiles of lactating bovine mammary tissue against nonlactating tissue on the BMAM microarray. Our results have yielded many novel and interesting genes exhibiting differential expression in lactating mammary tissue, including oncogenes (VAV3, C-myc), mediators of apoptosis (Caspase 8), and cell cycle regulators (LASP1).
- mammary development
- lactation
- expressed sequence tag
mammary gland development involves a series of complex physiological processes. There are six defined stages involved in development of the mammary gland including embryonic, prepubertal, pubertal, pregnancy, lactation, and involution. Each of these stages involves a complex interaction of hormones, growth factors, and signal transduction pathways, ultimately leading to expression of developmentally regulated genes. Although some factors involved in mammary development, such as the hormones prolactin, estrogen, and progesterone, have been known for some time, it has only been in the last 10 years that individual genes involved in mammogenesis have been identified. Gene targeting models have been used to uncover several important signaling pathways and genes that are directly involved in mammary development (for review see Ref. 22). For example, a functional Jak2/Stat5 pathway is absolutely required for mammary cell differentiation (35, 36, 44, 57). Additionally, the differentiation factor RANKL, which interacts with the Jak2/Stat5 pathway, is obligatory for alveolar development (14, 24). The cell cycle proteins cyclin D1 and p27 are also crucial for mammary development during pregnancy (13, 15, 18, 30, 45, 46 ,67).
Although these discoveries have greatly improved our understanding of the pathways and genes involved in mammary gland physiology, our understanding of this complex process is still far from complete. For example, target genes for the transcription factors involved in mammary gland development, such as Stat5, are still unknown. Furthermore, studies of cyclin D1/p27 have shown that there are additional, as yet unknown, cell cycle pathways involved (18, 21, 51). There is also very little understanding of how these different components interact with one another to coordinate the many physiological changes observed in developing mammary tissue. Clearly, a better understanding of the target genes and pathways involved in normal mammary development is needed before we can take a more focused approach to treatment and prevention of mammary gland diseases.
A good example of this is the relationship between lactation and breast cancer risk. It is known that breast-feeding greatly reduces the risk of breast cancer (5, 31, 47, 53, 69). The reason this occurs is not fully understood. Studies of apoptosis as it relates to normal lactation cycles and onset of breast cancer have led to some possible connections. Abnormal regulation of apoptosis has been demonstrated to be directly involved in human breast cancer (8, 20, 27, 34). Apoptosis also occurs during the processes of lactation and involution, utilizing many of the same apoptotic pathways that are involved in breast cancer development (for review see Ref. 20). Many of the genes that play a role in breast cancer apoptotic pathways have been identified (i.e., EGFR, IGF-I, bcl-2) using breast cancer cell lines. Although these studies have led to elucidation of some critical pathways in programmed cell death (PCD) related to breast cancer, the complex interaction of PCD pathways in vivo as well as their function in normal lactation cycles are unknown. A better understanding of the PCD pathways involved in normal lactation cycles and mammary development in vivo will be necessary before we can understand the link between protection from breast cancer and breast-feeding.
One of the models to study apoptosis during normal lactation cycles is the bovine. Because of their large size, cows provide a large source of lactating mammary tissue, which could be a limiting factor in other species. The cellular structure of the human mammary fat pad, parenchymal growth, and epithelial-stromal interactions are histologically similar to ruminants, but distinct from the mouse (23, 25). Despite differences in fatty acid synthesis in mammary cells of ruminants and nonruminants, the cellular similarities between ruminants and humans suggest that the bovine may prove to be a good physiological model along with rodents for human mammary physiology.
Bovine cell lines (including a bovine mammary epithelial cell line, MAC-T; Ref. 26) have been used successfully as model systems in a number of studies focused on host immune response (9, 32) and breast cancer (19, 48, 68). However, these studies were limited because, until recently, there has been a lack of attention in development and use of functional genomic resources in livestock animals. Functional genomics resources, such a high-density cDNA microarrays would allow for global analysis of gene expression from fresh tissues, providing a critical link to in vitro studies.
This paper describes development of a >1,000 gene cDNA microarray enhanced for bovine mammary transcripts (BMAM). To identify pathways and mechanisms specifically related to bovine mammary gland growth and development, gene expression patterns from eight different tissues including developing mammary tissue were compared using the BMAM microarray. We have identified a set of 16 genes that we believe to be preferentially expressed in the developing mammary gland. These genes may thus represent a developing bovine mammary gland gene expression "signature," providing good targets for future studies aimed at gaining a better understanding of mammary physiology. One of our long-term goals is to elucidate the genes and pathways associated with bovine lactation and involution and to use this as a model for human mammary gland development as it relates to human breast cancer risks. To begin this process, we compared gene expression profiles of lactating bovine mammary tissue against nonlactating bovine mammary tissue on the BMAM microarray. Our results suggest that many genes of interest exhibit differential expression in lactating mammary tissue relative to nonlactating tissue. In addition to the expected milk protein genes, genes involved in induction/repression of apoptosis, cell cycle control, and energy metabolism were also differentially expressed.
Selection of expressed sequence tag (EST) clones.
The United States Department of Agriculture (USDA), Agricultural Research Service, Beltsville Agricultural Research Center (ARS), previously constructed a cDNA library from pooled mRNA isolated from mammary tissues at eight physiological, developmental, and disease states (55). Additionally, there are currently available four pooled-tissue normalized bovine cDNA libraries constructed by the USDA Meat Animal Research Center (MARC) encompassing a wide variety of bovine tissue types, excluding mammary tissue (Table 1) (54). All ESTs in the MARC and BARC libraries are represented in The Institute for Genomic Research (TIGR) Cattle Gene Index (BtGI), and are ordered according to sequence overlap in clusters (TIGR Clusters, TC).
EST clones derived from mammary tissue were selected from available TCs using an "electronic subtraction" scheme. Based on the concordance of TC number and EST composition (TIGR file BTGI. TC_EST.01101), the library name (MARC or BARC) was determined for ESTs associated with a given TC through examination of the GenBank record for each EST. The TIGR BtGI (Release 5.0) contained a total of 20,209 TCs, 18,263 of which contained at least one MARC or BARC clone. All TCs containing a MARC clone were excluded, since these would have been derived from tissue other than mammary gland. When the 18,263 TCs were analyzed, 1,174 did not contain a MARC clone. If more than one BARC clone was available for a given TC, then the clone with the longest sequence read length was selected to represent each cluster through examination of the nucleotide sequence given in each clone's GenBank entry. Several Perl (v. 5.6.1) scripts were written to facilitate clone selection, and these are available upon request. Selected BARC clones (1,174) were used to form a bovine EST clone set (BMAM) that is enriched for transcripts derived from mammary tissue.
DNA sequencing and sequence analysis.
To make the BMAM EST clone set from the selected BARC clones, 1 μl of liquid culture from the selected BARC clones was transferred into individual wells of 96 well plates that contained 150 μl of Luria broth (LB) plus 8% wt/vol glycerol and 1,000 μg/ml ampicillin using a Tecan Genosys 150 liquid handling robot (Tecan US, Research Triangle Park, NC). Plates were covered with foil and grown overnight at 37°C with constant shaking at 225 rpm. Replicates of the BMAM library were made from the overnight cultures and stored at −80°C. A replicate of the entire BMAM EST clone set was processed for commercial high-throughput sequence verification (Genome Therapeutics). Genome Therapeutics uses the Amersham TempliPhi φ29 DNA Polymerase-based Rolling Circle Amplification kit (Amersham Biosciences, Piscataway, NJ) to amplify the plasmid prior to sequencing. Vector and quality trimmed sequence data from all of the BMAM clones were compared with the BARC EST clone set via BLAST. Of the 1,172 EST clones sent for sequencing, 1,114 matched (95%) with the original BARC clone sequence. This subset of 1,114 EST clones was selected to create the BMAM cDNA microarray.
Production of cDNA microarrays.
Clone inserts from the BMAM library were amplified by adding 1 μl of a 1:10 dilution [in double-distilled H2O (ddH2O)] of the TempliPhi φ29 Polymerase (Amersham Biosciences) reaction used for sequencing directly into PCR master mix [20 μM each dNTP, 0.5 μM forward primer, 0.5 μM reverse primer, 200 mM Tris-HCl (pH 8.4), 500 mM KCl, and 2.0 U of Taq DNA polymerase] in 96-well plates. The forward primer (5′ AATTCCCGGGATATCGTCGAC 3′) and reverse primer (5′ CCTCGAGGGATACTCTAGAGC 3′) are immediately adjacent to the insertion site for the cloning vector pSPORT6 used for construction of the original BARC EST library (55). Using the sequenced products as template for PCR amplification allowed us to ensure that the correct clones would be amplified. Following a preheat step of 94°C for 2 min, PCR was performed in a thermocycler (model PE 9700; PerkinElmer, Palo Alto, CA) using the following conditions: 94°C for 30 s; 60°C for 1 min, and 72°C for 3 min; for 30 cycles. Based on agarose gel electrophoresis of purified amplicons, our success rate with this protocol was >90%. Insert amplicons were purified using a Millipore Multiscreen PCR 96-well purification system (Millipore, Bedford, MA). PCR reaction products were added to corresponding wells of purification plates. The plates were placed on a Te-Vac vacuum manifold using a Tecan Genosys 150 liquid handling robot (Tecan US). A vacuum of 700 mBar was applied for 10 min to remove buffer and bind amplicon DNA. Final insert amplicons were resuspended in 25 μl of 50% DMSO for low-density (3,000–4,000 amplicons per slide) spotting on microarrays. Approximately 2 μl of each purified insert amplicon was separated on Invitrogen E-gel 96 1% agarose gels (Invitrogen, Carlsbad, CA) to ensure that amplicons representing each clone were represented on final microarrays in approximately equal concentration. A total of 5 μl of each purified amplicon was transferred to 384-well microarray source plates.
Microarrays were spotted using a GeneTAC G3 arraying robot (Genomic Solutions, Ann Arbor, MI) equipped with a 48-pin head, with each pin having a nominal end diameter of 200 μm. In 50% DMSO (low-density arrays), this arrangement yields spots of ∼250–300 μm. The pin configuration of the G3 yields microarrays consisting of 48 "patches" of spots. A microarray design was developed that allowed triple spotting of each clone within a patch and an overall 9×9 spot pattern in each patch (81 spots per patch). Additionally, each patch contains three GAPDH and three β-actin amplicons, which can be used for assessing RNA loading differences and spatial and potential dye effects. Three blanks surround one GAPDH gene spot, establishing a landmark for subsequent microarray image orientation and analysis. The blank spots can also be used to control for background effects on a global or patch-specific basis.
RNA extraction, labeling and hybridization.
Whole tissue samples of liver, spleen, thymus, adrenal, adipose, ileum, and lymph tissue were collected from a 3-mo-old Holstein steer at the time of slaughter. Whole tissue samples of mammary tissue were collected from two prepubertal Holstein heifers (136 kg and 227 kg in weight), a postpubertal Holstein heifer (310 kg in weight, not pregnant), and two lactating Holstein cows. The lactating Holstein mammary tissue for animal 1 was taken from a primiparous cow that was slaughtered at 181 days of lactation. The lactating mammary tissue for animal 2 was taken from a multiparous Holstein cow in its third lactation, with her last calf born 61 days before slaughter. Whole mammary gland tissues from five additional nonlactating and five additional lactating cows were collected for use in quantitative real-time PCR. The five nonlactating, nonpregnant Holstein heifers were all postpubertal and had slaughter weights of 291, 355, 336, 359, and 440 kg. The additional lactating mammary samples were from primiparous cows slaughtered at 181 days of lactation. A reference RNA for use in microarray experiments was prepared from an immortalized bovine macrophage cell line (56). We felt that a macrophage cell line would be a good source of cDNA that would not be expected to have a high level of gene expression against a panel of genes enhanced for mammary gland transcripts. Additionally, a stable cell line should be a reliable source of cDNA that could facilitate a connection between this study and future studies. For further experiments, we used the reference cDNA as a base to compare gene expression levels from various bovine tissue samples. The reference cell line was grown in 75-mm2 flasks containing RPMI-1640 with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2 and 95% air at 39°C. RNA was extracted from all tissues and cells using TRIzol reagent (Invitrogen) as described previously (59). Quantity and quality of total RNA extracted from all tissues and cells was estimated by UV spectrophotometry and electrophoresis on 1.2% native agarose gels. For all experiments, total RNA (8 μg) was used as template in reverse transcription reactions, using the Atlas Glass PowerScript Fluorescent labeling system (BD Biosciences, Alameda, CA) with oligo-(dT)18 as primer. Synthesis of cDNA in this system incorporates an amino-modified dUTP into the cDNA. Following first strand synthesis, cDNAs were labeled using n-hydroxysuccinate (NHS)-derivatized Cy3 and Cy5 dyes (Amersham Biosciences). Labeled cDNAs were purified to remove unincorporated dyes, combined and concentrated to ∼10 μl using Microcon 30 spin concentrators (Millipore). Prewarmed (70°C) SlideHyb no. 3 (Ambion), 100 μl, was added to probe mixtures, and the final probe solutions were applied to prewarmed microarray slides via the port of a GeneTAC Hybridization Station microarray hybridization chamber (Genomic Solutions). Hybridizations were conducted for 18 h using step-down temperatures from 65°C to 42°C in sealed chambers of a GeneTAC HybStation hybridization chamber. Following hybridizations, the station applies three washes, two with medium stringency buffer and one with high-stringency buffer (Genomic Solutions). Slides were finally rinsed briefly at room temperature in 2× saline sodium citrate and once in ddH2O. Washed microarrays were dried by centrifugation at 1,000 rpm in a cushioned 50-ml conical centrifugation tube.
Dried microarrays were scanned immediately using a GeneTAC LS IV microarray scanner and GeneTAC LS software (Genomic Solutions). GeneTAC Integrator 4.0 software was then used to process array images, align spots, integrate robot-spotting files with the microarray image, and to export reports of spot intensity data. The final report was retrieved as raw spot intensities in comma separated value files.
Microarray data analysis.
Microarray data was normalized using the scatter plot smoother LOWESS (10) from the statistical package SAS ("PROC LOESS") according to the method of Yang et al. (64). Briefly, the normalization process is as follows, M-A plots are constructed for each slide, where log-intensity ratios M = log(Cy3/Cy5) = [logCy3 − logCy5] are plotted against the mean log-intensity A = [(logCy3 + logCy5)/2] as described by Yang et al. (64). Normalization is then performed considering a robust local regression technique (11), using the LOWESS procedure of SAS (SAS Institute, Cary, IN). Efficiency of LOWESS normalization is assessed by monitoring M-A plots before and after normalization. The normalized data is then back transformed prior to further statistical analysis using the formula where logCy3* and logCy5* are the normalized log intensities. Here, M* = M − M^ and represents each of the normalized M values, with M^ being the LOWESS predicted value for each spot.
LOWESS normalized array data was imported into Microsoft Excel for further analysis. After back-transforming normalized intensity values as above, the median blank intensity on a microarray for each dye was subtracted from the respective normalized spot intensity values. Residual intensity values were again log transformed prior to further analysis. Log-transformed and blank subtracted values were used to calculate a mean log expression difference for each gene for each tissue compared with reference cell cDNA. Since each gene is technically replicated three times on an array, a P value (t-test) can be determined for evidence of differential expression on each gene. This P value strictly refers only to the particular sample vs. reference rather than a biologically replicated mammary vs. reference comparison for any given gene on the array. The antilog of the mean log expression difference for an individual gene on the array yields the approximate fold change in expression in this gene between the tissue of interest and the reference cDNA. For a general mammary vs. reference comparison, the average log difference between the tissue's corrected intensity value and the reference RNA corrected intensity value was used.
By performing experiments that hybridized the same sample labeled with both Cy3 and Cy5 on the same slide, we were able to empirically determine the rate of false-positive signals expected on the BMAM cDNA microarray. By determining the false-positive rate we have a measurement of the rate of truly null features being called significant. Additionally, a conservative implementation of the false discovery rate (FDR) was calculated on individual microarray experiments (6). The FDR gives the rate that significant features are truly null. For a gene to be considered as significantly different, the FDR P value was required to be ≤0.1. This liberal cutoff was used to avoid declaring too many false negatives, which may be as serious as false-positive declarations in genomic studies.
Microarray experiments.
A reference microarray experimental design (28, 29, 65) was used to compare prepubertal mammary gland (136-kg Holstein heifer) gene expression against a panel of seven other tissues (experiment 1). This was accomplished by first starting with the 44 genes that had significantly greater expression in developing mammary tissue compared with reference cells (fold change, >1.5; FDR: P ≤ 0.1). The seven other tissues' gene expression levels for these 44 genes relative to reference (log fluorescence intensity of the tissue's cDNA minus the fluorescence intensity of the reference cells within the corresponding arrays) were subtracted from the developing mammary gland's log fluorescence intensity (also expressed as a deviation from the fluorescence intensity of the reference cells within the corresponding arrays). Following the subtraction, the antilog of the difference was used to calculate the fold difference between the tissue and mammary gland. This comparison was done for each of the seven tissue types against the prepubertal mammary gland.
Experiment 2 compared animal 1 vs. nonlactating mammary tissue (136-kg Holstein heifer) and experiment 3 compared animal 2 vs. nonlactating mammary tissue (227-kg Holstein heifer). Mean log expression differences from animal 1 vs. nonlactating (136-kg Holstein heifer) (experiment 2) and mean log expression differences from animal 2 vs. nonlactating (227-kg Holstein heifer) (experiment 3) were used as biological replicates. An overall average log difference between lactating and nonlactating tissue was calculated using the mean log difference for each gene for animal 1 and animal 2. A P value (t-test) for differential expression was based on these two averages. Data for the 13 microarray experiments used in this report can be found in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO), series accession number GSE279.
Real-time quantitative PCR.
To confirm gene expression differences observed from microarray results, quantitative real-time PCR was performed on six genes with overexpression in lactating mammary tissue relative to nonlactating tissue (based on experiments 2 and 3). These six genes were chosen because they had a known function that has not been previously associated with bovine lactation. To better account for biological variation among the samples, a total of seven nonlactating mammary samples (two prepubertal samples used in the microarray experiments and five additional postpubertal nonpregnant mammary samples) and seven lactating mammary samples (two lactating samples used in the microarray experiments and five additional midlactation mammary samples) were used in real-time quantitative PCR. The six genes were BMAM_BE589961 (Caspase 8), BMAM_BE476665 (CD47 antigen/integrin-associated protein), BMAM_BF868293 (LIM and SH3 protein 1), BMAM_BE588590 (PBK1), BMAM_BF230907 (Vav 3 oncogene), and BMAM_BE481095 (c-myc proto-oncogene).
Real-time quantitative PCR primers were designed using Primer Express ver. 2.0 (Applied Biosystems, Foster City, CA) using the sequence of the respective EST as the source DNA. Primers for the six genes used are BMAM_BE589961 (forward, 5′ ACTGGATCTGCTGCTTAACTACTTGTAT 3′; reverse, 5′ GCCCTGCCGGGTATCTG 3′), BMAM_BE476665 (forward, 5′ TGTACCTTTTAATATGTAGTCAGCTCTCATT 3′; reverse, 5′ AGGGACTGCCTCCAAAGACA 3′), BMAM_BF868293 (forward, 5′ GAGGAAGGGAAAGATGGTGCTA 3′; reverse, 5′ AATCAGGCAAGGGTGGTCATAG 3′), BMAM_BE588590 (forward, 5′ CTGAAGCAGGCTGCAAAGG 3′; reverse, 5′ GGAACACCCCCAGTTTTGG 3′), BMAM_BF230907 (forward, 5′ CGCGTCCATCCCATACTGTT 3′; reverse, 5′ GGGATGAGGTAGAGGAGAGCAA 3′), and BMAM_BE481095 (forward, 5′ GCGCAAAGAGGATTCGTCTCT 3′; reverse, 5′ GCGCCTCTCTCTCGGAGTT 3′).
There was not publicly available a full-length cDNA sequence for bovine β-actin; therefore, the BMAM clone BMAM_BG689033 (596 bp) that has significant BLASTN similarity to human and mouse β-actin (E value <10−50) was sequenced in its entirety. The entire length of clone BMAM_BG689033 was 1.8 kb and contained the full-length cDNA sequence for bovine β-actin (GenBank accession no. AY141970). Real-time quantitative PCR primers were designed using Primer Express ver. 2.0 (Applied Biosystems) based on the bovine β-actin sequence (forward, 5′ CGCCATGGATGATGATATTGC 3′; reverse, 5′ AAGCCGGCCTTGCACAT 3′).
Quantitative PCR was performed in duplicate for each sample on an ABI 7000 Sequence detection system (Applied Biosystems) using the default two-step amplification procedure and 2× SYBR Green Master Mix (Applied Biosystems) in 50 μl reaction volume with 300 nM of each primer and 50 ng of cDNA.
Relative quantitation of mRNA abundance for genes of interest were calculated using the 2−ΔΔCT method of Livak and Schmittgen (37). Amplification of β-actin from each sample was used for normalizing quantitative PCR data (37). Relative quantitation of genes from individual lactating mammary gland samples was compared with mean data from nonlactating tissue samples.
An overall average between lactating mammary samples and nonlactating mammary samples was calculated by taking the average of the relative concentration of each lactating mammary sample compared with the overall nonlactating mammary samples. A P value (t-test) for the overall average was established using these seven averages. Thus these P values appropriately take into account true biological replication.
BMAM EST characterization and database.
A web-accessible resource was established (http://nbfgc.msu.edu) to provide information on all EST clones contained in the BMAM microarray. The web database can be searched by cDNA clone name, ontology, or keyword (keyword based on BLAST information from the cDNA sequence). Clones in the BMAM EST collection were functionally classified using GeneSpring ver. 4.1.5 (Silicon Genetics, Redwood City, CA) Build Simplified Ontology function, which is based on the Gene Ontology Consortium classifications (2). Ontology classification assignment for each clone is based on the description line of the top BLASTX hit if available; otherwise, it is based on the top BLASTN hit corresponding to a functional gene ontology. Ontological classification for each EST is web-linked to AmiGO!, an HTML-based browser for The Gene Ontology from the Gene Ontology Consortium (http://www.geneontology.org). Of the original 1,174 BMAM EST clones, 733 (62%) matched to known genes (based on BLASTN or BLASTX matches with E value < 10−15). Of these 733 clones, 116 matched to characterized bovine genes; the remaining 617 ESTs matched to known genes from other species. In total, 246 of the 733 clones had matches to hypothetical genes or genes with no known function in either bovine (18), human (225), or mouse (5). We assigned 383 of the 773 genes with putative gene identifications to one of the 3 major categories of ontology (biological process, cellular component, and molecular function) using GeneSpring ver. 4.1.5 (Silicon Genetics).
A wide range of functional ontology categories are represented in the BMAM EST collection (Fig. 1). A large proportion of signal transduction, transport, and mitochondrial related genes were identified, consistent with the many diverse functions in growth, differentiation, milk synthesis, immunity, and involution characteristic of the mammary gland. The BMAM EST clone collection also contains 11 caseins and casein kinase related genes, as well as many plasma membrane associated genes that may be highly expressed in development of mammary parenchymal tissue, basement membrane, and extracellular matrix (ECM). Since the BARC library is a pooled library from mammary gland at different physiological and developmental states (55), these genes may be expected to play a role in developing as well as lactating mammary gland.
BMAM microarray characterization.
The BMAM microarray contains a total of 3,888 spots. These consist of the 1,114 BMAM EST clones spotted in triplicate within a patch, 198 spots with Bos taurus GAPDH cDNA, 150 spots with B. taurus β-actin cDNA spots, and 198 blank spots. Triplicate spotting of the BMAM EST clones improves reproducibility and allows for a statistical partitioning of measurement from experimental error within an array.
As a first step in ascertaining expression patterns of genes represented on the BMAM cDNA microarray, it was important to determine the potential for false-positive expression changes. The false-positive rate was assessed by hybridizing differentially labeled aliquots of cDNA from the same sample of reference RNA. This process was replicated to produce two separate microarrays. Since the Cy3- and Cy5-labeled samples on these microarrays were from identical cDNA sources, there should be no significant differences in gene expression, and we would anticipate microarray analysis to yield equal fluorescent intensities for both the Cy3 and Cy5 labels at every spot on the microarray. When data from these microarrays were combined and analyzed, there were in fact, few differences between LOWESS normalized Cy3 and Cy5 intensities for most genes. A total of two genes showed a >1.4-fold expression difference, however, neither of these expression differences were statistically significant at FDR (P ≤ 0.1 across the two microarrays). This result demonstrated that design of the BMAM microarray, combined with LOWESS normalization and blank subtraction yields a low rate of false positives. Using a threshold of >1.5-fold expression difference allows us to approximate the false-positive error rate as <0.1%, of all amplicons showing differential expression when their actual expression levels should be similar. Therefore, for all subsequent experimental comparisons a threshold of 1.5-fold change was used as a cutoff, combined with a significance between gene replicates of FDR P ≤ 0.1.
Defining a set of genes expressed in prepubertal mammary gland.
We wished to define a set of genes preferentially expressed in the developing mammary gland. To accomplish this, we first performed an experiment in which cDNA derived from prepubertal mammary tissue (136-kg Holstein heifer) was compared with cDNA from the reference RNA cells on the BMAM microarray. There were 44 BMAM EST clones that had a significantly greater expression (fold change, >1.5; FDR, P ≤ 0.1) in prepubertal mammary tissue and 1 EST had a significantly greater expression (fold change, >1.5; FDR, P ≤ 0.1) in reference cells relative to prepubertal mammary gland. This result demonstrates our ability to detect gene expression in developing mammary tissue and that, as expected, gene expression in the reference cells was low compared with mammary tissue.
Based in part on our preliminary analysis, we hypothesized that the BMAM cDNA microarray could be used to establish a gene expression profile in developing mammary gland. Specifically, we were interested in identifying genes that would have a high probability of being preferentially expressed in the prepubertal mammary gland. To accomplish this, an experiment was performed (experiment 1) to compare gene expression levels of several tissue types (adipose, liver, adrenal, lymph, spleen, thymus, and ileum) to developing mammary tissue by utilizing a reference microarray experimental design (28, 29, 65) In this case, cDNA from the various tissue types was directly compared with cDNA from reference cells, using separate microarrays for each tissue. Of the 44 genes with >1.5-fold expression in prepubertal mammary vs. reference, 39 had >1.5-fold higher expression in prepubertal mammary relative to adipose. Of the remaining 39 genes, 35 were also expressed more than 1.5-fold greater in prepubertal mammary gland than in liver, and so on (Fig. 2). When all of the data were combined and analyzed, 16 genes (Table 2) exhibited greater expression (>1.5-fold) in developing mammary gland than in the 7 other tissues types examined. These 16 genes did not show any significant difference in expression when RNA from prepubertal and postpubertal mammary tissue were directly compared on the BMAM microarray (overall mean expression ratio 0.95) and when RNA from prepubertal (136-kg Holstein heifer) and lactating mammary were directly compared with lactating mammary tissue (overall mean expression ratio 1.05).
Defining a set of genes differentially expressed in lactating mammary tissue.
Our next objective was to find genes that are differentially expressed in lactating mammary tissue relative to nonlactating tissue. To accomplish this, we performed microarray experiments directly comparing RNA from nonlactating mammary tissue (136-kg Holstein heifer and 227-kg Holstein heifer) to RNA from mammary tissue of two midlactation cows (animal 1, animal 2) (experiments 2 and 3). Using a cutoff of ≥1.5 fold difference in gene expression at FDR P ≤ 0.1 in lactating tissue compared with nonlactating tissue, there were a total of 75 genes upregulated in lactating mammary tissue from animal 1 relative to nonlactating tissue (136-kg heifer), and 112 genes upregulated in lactating animal 2 relative to nonlactating tissue (227-kg Holstein heifer). As excepted, genes encoding αS1-casein and β-casein were among those with enhanced expression in lactating tissue from both cows relative to nonlactating tissue. A total of 77 genes for animal 1 and 141 genes for animal 2 were expressed significantly lower (less than or equal to a −1.5-fold difference, FDR P ≤ 0.1) in lactating mammary tissue relative to nonlactating tissue.
To establish the biological significance of differential gene expression observed in nonlactating vs. lactating microarray comparisons, expression data from the two lactating cow samples were combined and analyzed. Genes that had a ≥3-fold overall overexpression in lactating mammary gland relative to nonlactating are included in Table 3. Genes that had ≤3-fold overall repression in lactating mammary tissue relative to nonlactating are included in Table 4. There was large variability in some genes because the magnitude of expression differed between cows, even though the direction of expression was the same. Due to this large variability, real-time quantitative PCR was performed on selected genes to confirm microarray results.
Quantitative real-time PCR.
Figure 3 depicts the average relative amount of mammary mRNA for six genes with overexpression in lactating mammary tissue relative to nonlactating tissue (based on experiments 2 and 3) from seven lactating animals compared with seven nonlactating mammary samples using quantitative real-time PCR (36). Five genes were highly significant (P ≤ 0.05) and had >2.5-fold expression in lactating mammary tissue relative to nonlactating (C-myc, Caspase 8, VAV3, LASP1, and PBK1, Fig. 3). CD47 had 3.5-fold expression in lactating mammary gland relative to nonlactating mammary gland (P = 0.09, Fig. 3).
A cDNA microarray resource for studies of bovine mammary gland physiology has been developed and tested for its utility in detection of differential gene expression during physiologically relevant points in mammary gland development. The BMAM EST clone set and microarray contain a directed set of clones enhanced for transcripts found in bovine mammary gland and is annotated in a web-accessible database (http://nbfgc.msu.edu) designed to assist in interpretation of microarray data. BMAM clone data in our database allows retrieval of the top BLAST results for each clone against sequences in GenBank nonredundant (nr) database. Additional information, including clone sequence data, the TIGR TC number, and gene ontology information are also displayed. Links to public databases, such as GenBank, TIGR BtGI, and the Gene Ontology Consortium's AmiGO! allow rapid retrieval of associated information for each clone. The database is searchable with regards to clone name, keywords derived from BLAST hits, BLAST searching within the database, and ontology term. Search results are of primary importance when analyzing data from microarray experiments, allowing for easy integration of gene expression data and putative gene identification and ontology information of the transcript represented by each clone.
We assessed the ability of BMAM microarrays to detect gene expression in developing mammary gland. In total, 44 genes displayed greater expression in prepubertal mammary gland relative to reference cells. Expression of RNAs represented on the BMAM cDNA microarray were further evaluated in a group of seven tissues (adipose, liver, adrenal, lymph, spleen, thymus, and ileum) using a reference design, allowing for cross-microarray comparison. After comparing gene expression data from all 7 tissues with prepubertal mammary gland, 16 genes were identified as having a high probability of being preferentially expressed in developing mammary tissue. For example, expression of butyrophilin (Table 2), a plasma membrane protein involved in discharge of milk fat globules (4), would be expected particularly during lactation, although a role may also exist in the developing mammary gland. Preferential expression of another 10 of these genes in prepubertal mammary gland is reasonable given their indicated roles as integral membrane proteins in cell proliferation and in other functions likely occurring in tissue that is growing and differentiating. The fact that 5 genes of the 16 genes (BMAM_BG691636, BMAM_BE486522, BMAM_BE486043, BMAM_BE477209, and BMAM_BE478128, Table 2) in this group have an unknown function provides an indication that there is much to learn despite significant prior research on control of mammary gland development and function in humans, mice, and cattle.
The fact that only 16 genes were detected with expression highly specific to developing mammary gland may, in part, be due to the relatively recent evolutionary acquisition of mammary glands in mammals. It has been suggested that many, if not all genetic pathways involved in development of the mammary gland are used in tissues that appear earlier in evolution (21). Genes regulating mammary gland development have been gained from a wide variety of tissue types, as illustrated by genes known to be involved in mammogenesis, such as Jak2/Stat5, RANKL, and the cyclins. These genes are also known to regulate bone morphogenesis, reproduction, retinal development, and brain and other tissue developmental programs. Despite this, the 16 genes identified in the current report appear to be highly specific to developing mammary gland and as such are potentially very good candidates for playing an important role in regulating mammogenesis.
Similarly, in a comparison of gene expression profiles in cDNA derived from lactating and nonlactating mammary tissue RNA on the BMAM microarray, we were able to identify numerous interesting genes that are affected by lactation in either a positive or negative manner (Tables 3 and 4). Many of the genes identified have not been previously associated with lactation. For example, LASP1 has 18-fold higher expression in lactating mammary tissue relative to nonlactating tissue as determined by microarray analysis (Table 3). LASP1 was first identified from a breast cancer-derived metastatic lymph node cDNA library (58). LASP1 has subsequently been mapped to the long arm of human chromosome 17 (17) and mouse chromosome 11 (49), which is almost completely syntenic with bovine chromosome 19 (3). Chromosome 17 in human is also where the breast cancer related genes c-erbB-2 and BRCA1 (43) are located. It has been suggested that c-erbB-2 and LASP1 belong to the same amplicon due to the co-amplification and co-overexpression of both genes in 12% of mammary tumors (7). LASP1 may be involved in tumorigenesis and/or in tumor progression (7). Schreiber et al. (50) showed that LASP1 is expressed in various degrees in a wide variety of adult mouse tissue, including mammary gland (some of the tissues where there was the strongest expression, such as placenta and colon were not included in the MARC cDNA libraries, which may explain why LASP1 was included in the BMAM EST collection). The strong evolutionary conservation, actin binding domain, and ubiquitous expression of LASP1, suggests that this gene plays an essential role in basic cellular functions (50). Lin et al. (33) have demonstrated that LASP1 is a downstream cytoskeletal target of c-ABL that functions to prevent cell migration on the ECM. Marti et al. (39) showed that there was a weak induction in actin during involution compared with lactating tissue. The fact that LASP1 is overexpressed in lactating mammary gland may be related to its function in regulating cell migration via its interaction with the actin cytoskeleton.
It is interesting to note that there is increased expression during lactation of one of the initiators of apoptosis (Caspase 8, 24-fold greater expression relative to nonlactating mammary tissue, Table 3), and two oncogenes, VAV3 and C-myc, (>10-fold higher in lactating tissue relative to nonlactating tissue, Table 3). Both oncogenes are regulators of cell division. Although the occurrence of apoptosis is well established during involution of mammary gland in cattle (63) and mice (38, 39, 40, 41, 59), few studies have addressed the significance of apoptosis in normal lactation. One study using immunohistochemistry to analyze expression of apoptosis-related genes in lactating goat mammary tissue (62), showed an increase in expression of many apoptosis-related genes over the course of lactation. Activated genes included TGF-B1, TGFBIIIR, Bcl-2, and caspase 3. Since VAV3 is regulated by TGFB (61), our results provide preliminary evidence that PCD may also be involved during lactation in cattle.
In addition to known genes discussed previously, we also identified five genes that have a greater than fourfold higher expression in lactating mammary tissue relative to nonlactating tissue that do not match any known genes (Table 3). For example, BMAM_BG689327 had >24-fold higher expression in lactating mammary tissue compared with nonlactating mammary tissue (Table 3). Further characterization of these genes and their protein products, as well as their role in mammary physiology is clearly warranted.
Glyoxalase I (GLO1) was significantly repressed (greater than 3-fold, Table 4) in lactating mammary tissue relative to nonlactating mammary tissue. GLO1 is a detoxifying enzyme, which has elevated expression in human breast cancer tissue compared with normal tissue (49). GLO1 is also abundantly expressed in bladder and prostate cancer tissue (12, 42) and has been implicated in multidrug resistance in tumor cells (10). There has been no previous association of GLO1 with lactation physiology.
The milk proteins comprise a majority of the proteins expressed in lactating mammary gland (for review see Refs. 1 and 16), so clearly transcripts encoding the caseins would have the highest absolute expression in lactating mammary tissue. The caseins (αS1 and β) and the mammary cell-specific proteins of the whey fraction (lactalbumin and lactoglobulin) had greater relative expression in lactating mammary tissue compared with nonlactating tissue (Table 3). Because of the difference in fold change between animal 1 and animal 2 relative to nonlactating tissue (4.5 and 11.7, respectively, Table 3), αS1-casein had a P value for the combined data of 0.14 (Table 3). We acknowledge a limitation of the present experiment was biological variation from the limited sample size and heterogeneity in time of collection during lactation. However, for the individual microarray experiments, αS1-casein has the highest significance level (animal 1, FDR P = 4.3 × 10−05; animal 2, P = 5.6 × 10−09) for the genes that have increased expression in lactating tissue relative to nonlactating tissue. These expected results lend credence to the overall gene expression patterns observed in this microarray study.
To confirm gene expression data observed in lactating and nonlactating mammary tissues on the BMAM microarray, expression of six selected genes was tested with quantitative real-time PCR. All six had similar gene expression profiles in quantitative real-time PCR assays compared with microarray studies, although the level of expression varied between samples (Fig. 3). There was also variability in level of expression between lactating samples, although the trend was in agreement with microarray results (Fig. 3). This variability could be due to heterogeneity in cell type (since whole tissue samples were used), variation between animals (since samples were not strictly age matched), or to the outbred nature of animals used in this preliminary study. Regardless of the source of variability, it is clear that the trend is upregulation of CASP8, CD47, c-MYC, LASP1, PBK1, and VAV3 in lactating mammary gland vs. nonlactating mammary gland. Future work is needed to further elucidate gene expression patterns during the full course of mammary development and lactation cycles.
In summary, we have designed an EST and microarray resource enhanced for transcripts derived from bovine mammary tissue (BMAM). This resource should prove valuable in functional genomics studies of differential gene expression in the bovine mammary gland. We have preliminary gene expression patterns associated with mammary gland development and have identified 16 genes comprising a potential developing mammary gland gene expression "signature." These genes may prove to be useful targets in studies of hormonal and metabolic regulation of mammary development. Analysis of differential gene expression between nonlactating and lactating mammary tissue revealed the identities of several previously unknown genes that may play important roles in the bovine mammary gland during lactation. Further characterization of these genes and their protein products should improve our understanding of mechanisms important in mammary physiology. In addition, the BMAM microarray, combined with other bovine-specific microarrays developed by our group (66) should help advance in vivo studies aimed at shedding light on mechanisms that underlie diseases of the mammary gland, such as mastitis and cancer.
We gratefully acknowledge the excellent assistance of Guilherme Rosa, Department of Animal Science, Michigan State University, in statistical analysis of microarray data. We also acknowledge the excellent support of Peter Saama, Department of Animal Science, Michigan State University, in maintaining the Center for Animal Functional Genomics website and database.
Work presented in this report was supported by the Michigan Agriculture Experiment Station, the Office of the Vice President for Research and Graduate Studies at Michigan State University, and by USDA Initiative for Future Agriculture and Food Systems (IFAFS) Grant 2001-52100-11211.
↵1 This article was submitted for review in response to a Call for Papers on Comparative Genomics.
Address for reprint requests and other correspondence: S. P. Suchyta, B215 Anthony Hall, Center for Animal Functional Genomics, Michigan State Univ., East Lansing, MI 48824 (E-mail: suchytas{at}msu.edu).
- Copyright © 2004 the American Physiological Society


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The two were broken by the time they reached the possessed Lord.
It became a battle of wits, and Link was victorious. Yet fear ran through his veins. Countless times the boy had fought sword in hand, won and continued. Soon it seemed as if his entire life revolved around saving the Princess Zelda but when the day finally came, Link wasn't focussed. A piece was missing, and it was huge, frightening monstrous.
Except pieces didn't scream.
A Knight has nothing to protect without a Queen.
Her absence created an effect, and he was desperate to touch her. However fate seemingly wasn't on his side when Ganondorf closed a barrier between them. A horrible rage ignited within him, and the barrier didn't fade, unlike his pride. For it was as if the barrier was not created by only Ganondorf, but everyone around him.
Never will Link reach her. They simply weren't.
Suddenly everything became nothing.
When they were reunited, it lasted less than an hour. Such short little time provided by the Devil who grinned at the two, set his plans forwards.
Link knew his responsibilities were to protect Hyrule, but to also guard the only woman who stood by him throughout. Adrenaline pumped furiously, and he shuddered, desperate to win, to create peace.
Of course the Knight was not expecting a betrayal.
It was her eyes.
Dark and sad, but expressing power which she refused to claim her mind.
The familiar sensation of being warped tingled his skin, and all he could think of was the barrier again, how they were separated, refused to belong.
His shadow, his companion...
His friend, his reason...
His Princess.
Before anymore words could escape his lips Link vanished and became the air, vanishing from her sight, a very peculiar and wrong circumstance.
It felt as if his heart were ripped out of his chest and trod on.
His entire vision clouded in agony and there wasn't any pain which compared to this.
The Fused Shadows, the helmet, the crown were held up by the man he loathed bitterly, and the gaping hole in his chest was soon filled with furious anger. It was mocking, a horrible, horrible feeling. And the crown broke, crumbled to the ground, useless.
How it resembled his heart, their fate, his loss.
For his enemy, his motive, his reason, his heart were gone.
There is nothing left.
A warm hand touched his wrist.
Link clenched the blade.
The Queen may have lost the battle,
But a King fights to the bloody end.
Crown | Link and Midna by Bureizu-za-VampireWatch
©2012-2017 Bureizu-za-Vampire
Have some Link/Midna angst. It's mostly metaphorical gobble.


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Cholene in the news
Am I allowed to say, "you go girl!"? If not, forget I ever said it.
Gay Hurricane Katrina hero speaks
Larry Buhl, PlanetOut Network
Monday, July 24, 2006 / 02:37 PM
Cholene Espinoza admits that she is always on a mission. More than a dozen years ago, she was the second woman to fly the U-2 reconnaissance aircraft in the U.S. Air Force. In 2003, she took a job as a military correspondent for Talk Radio News Service and was embedded with the U.S. Marine Corps First Tank Battalion in Iraq.
Now her mission is rebuilding a church and community center devastated a year ago by Hurricane Katrina. Since September, Espinoza and her partner, Ellen Ratner, have made numerous trips to Harrison County, Miss., and helped raise more than $600,000 for a community education center there.


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Though parents may want to postpone the dating years indefinitely, a teen will likely fall in love with one of his peers some day. Parents may find themselves wondering if a teen's new romance is love or a fleeting crush. While you may not approve of your teen's other half, understanding the science of love may help you appreciate your teen's feelings.
Why Do Teens Fall In Love?
There are several factors that may cause us to become attracted to someone, and then to eventually fall in love. Teens may find one another not only physically attractive, but your teen may be attracted to someone's voice or what they have to say, according to At-Bristol, a science center in England. It is possible that teens are, at least on a subconscious level, also deciding if they would like their future children to have a potential mate's genes, according to the BBC. Researchers are also investigating the possibility that pheromones, odorless chemicals humans produce, may also play a role in human attraction.
Hormonal Changes While In Love
During the initial stages of love, a teen's body is flooded with testosterone and estrogen, according to the Orlando Science Center. During the second stage of love, your teen may experience an increase in hormones like dopamine, serotonin and norepinephrine. During the last stage of love, your teen's oxytocin and vasopressin levels increase.
Effects Of Hormonal Changes
The first stage of love, when testosterone and estrogen levels rise, prompt your teen to start looking for a potential mate, according to At-Bristol. As your teen enters the second stage of love, she may show classic signs of obsession, like spending all of her time with her new boyfriend or pining for him when they are separated, according to the BBC. Sleeplessness may increase and appetite may decrease during this stage. During the final stage of love, attachment and bonding increase, according to KidsHealth.org. Vasopressin and oxytocin work to keep humans bonded and in love over the long term.
Additional Information
During the later teen years, love can be a serious commitment like that seen in adults, according to KidsHealth.org. During the earlier teen years, love may be more about having fun and getting to know someone else, or it could be a status symbol to show off to peers.


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ROCHESTER — DeNiro Development won the Rochester Men's Hockey League championship last Thursday (March 13), beating RSA Realty, 4-3, in the third and game game of the best-of-three championship series at the Rochester Arena.
DeNiro (13-7-5) jumped out to a 3-0 lead with a goal injt he first period and two in the second, before RSA (16-5-3) came back with three goals of its own to tie the game at 3-all.
DeNiro's Matt Watson scored the game-winner at 5;20 of the third period from Lyle Marelli.
DeNiro led 1-0 after the first period on Watson's first goal at 3:33 from Tom Breslin.
DeNiro upped the margin to 3-0 in the second on goals by Tom Gillum (from Josh Perry and Jordan Blair) and Gillum (from Perry).
RSA then struck for two goals 26 seconds apart to close out the third down 3-2. Jeff St. Laurent scored on the power play from Peter Keilty, and Brian Donovan Jr. followed with a score from Derek Ceppetelli and Ryan Montecalvo.
At 3:59 of the third, Montecalvo tied the game up with Donovan Jr. getting the assist.
Derek Colwell had 33 saves for DeNiro. Casey Cox stopped 30 shots for RSA.


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JUSTIN Burnell is keeping positive about London Welsh's chances of qualifying from Pool 2 in the British & Irish Cup.
Welsh opened up with narrow defeats to London Scottish and Pontypridd before hammering Edinburgh Academical, who they visit today, 73-10.
Another big win for Welsh and a victory for Scottish at home to Pontypridd would put Burnell's men back in the picture, with only the pool winners progressing.
"It's up to us," said Burnell.
"If we manage to get another good performance against Edinburgh and win, we can still be in a good position.
"Pontypridd have got to come to us and they won't relish that.
"We need London Scottish to beat Pontypridd and that would create an opportunity for us."
First, however, Burnell is not underestimating today's opponents.
"Edinburgh were a bit disappointed with their own performance," he said.
"They are going to come with a totally different approach and intensity. They are at home and it means a lot to them."
Reflecting on last week's victory, Burnell said: "I just thought our attitude was good.
"When we had the chance to create, we scored ten tries."
And having scored freely in the home match, Burnell will be looking for more of the same attacking today.
"If it's not broken, there's no need to fix it," he said.


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Life has been hectic around here lately. Between spring cleaning, spring fever, sports, after school activities, traveling and the upcoming Flowertown Festival, I haven't had a chance to catch my breath. Let alone catch up with my girl friends.
So I decided to have a Girls' Night In. I didn't feel like putting on anything but yoga pants, let alone go out on the town, so I invited the ladies in my Bible Study to have a Spa Night at my house on Monday. I'm not sure if you've ever had one, but Girls' Night In is the best. It's just about some good food, good drinks, and good friends.
Now, I love to entertain, but I just wanted to relax for this party, so I invited my friend Allison over to pamper us with some of her awesome Arbonne products.
1. Good food. Have a mix of healthy, sweet and savory things to eat. I like to make everything finger foods if possible to make nibbling easier. For this evening party, I had pound cake cubes, a fruit platter with whipped cream, and a cheese cube and mini club cracker platter with honey. I found colorful spring plates and napkins to complete my buffet. Toothpicks replaced silverware!
2. Good drinks. Keep in mind the tastes of the guests at your party. At this particular one, I chose to serve chilled white wine and homemade Skinny Sangria. I found the recipe over at Living Locurto if you want to try it. Some said it was too sweet, but most of us loved it! Low on carbs and sugar…
3. Good SWAG. You know me. I am all about some free stuff. So when I have parties, I love to give out little favors, or have a little something that my guests can take home. When I threw my Pinterest project party, we all made a reclaimed wood sign to take for our homes. When I threw Millie's Mermaid birthday party, the little girls got to go home with a silky pearl hair clip. For this party, spring was in the air…so the takeaway was little pots filled with gardening tools, seeds, and {of course} some Arbonne samples.
4. Good Friends. I'm blessed to be surrounded by many wonderful people that I can call friends. I have a group at church, a group in my mom's club, a group in my neighborhood, etc. So when I plan my Girls' Night In nights, I try to stick to one group at a time to keep it intimate. That way everyone pretty much knows everyone else and can feel comfortable and relax.
5. Good Entertainment. Whether you are watching the latest {or a classic} chick-flick, applying fun Jamberry Nail wraps, or even just laughing around a campfire, there needs to be a focal point to the gathering. On Monday, it was a spa night courtesy of Arbonne. Allison has us all wash our faces and apply a Seasource Sea Mud & Body Mask to our faces….which appropriately turned us green for St. Patty's Day pics on Instagram!
While our face masks were working their magic, our feet were soaking in buckets of warm water and Aromassentials Unwind Bath Salts. Glass pebbles from the dollar store at the bottom of the buckets gave our feet a nice massage as they soaked, and we scrubbed Awaken Sea Salt Scrub to exfoliate. My feet were so soft even the next day, which worked out well since I had a podiatrist appointment for my Plantar Fasciitis!
We also got to try out a ton of facial products after we took off our masks, including some spray toner {so you don't have to waste cotton balls}, instant lift gel, and an intensive renewal serum…all of which tightened, toned and smoothed our faces. After all the pampering, my face felt and looked fantastic!
Giveaway Time!
Today I've got a fabulous Arbonne Giveaway worth $87. Its the Complexion Perfecting Set, which includes a Purifying Cleanser + Toner, an Oil-Absorbing Day Lotion Broad Spectrum SPF 20 Sunscreen, and a Mattifying Powder.
The Purifying Cleanser + Toner cleanses and tones, visibly reducing oiliness for refreshed-looking skin.
The Oil-Absorbing Day Lotion Broad Spectrum SPF 20 Sunscreen helps absorb oil and provides sun protection, leaving skin with a fresh, matte appearance.
The Mattifying Powder takes away unwanted shine, while reducing the appearance of redness for a perfect, matte finish. It dusts on lightly over or under moisturizer or makeup.
This giveaway ends at Midnight on Thursday, March 26th.
Good Luck!


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Tigers' season ends in game 2 of sub-section tournament
Morris Area was knocked out of the Section 6A baseball tournament by No. 1 seed Wheaton/ Herman-Norcross Thursday at Chizek Field.
Morris Area scored the first run of the game, but the Warriors plated the rest to advance in the tournament with an 11-1, six-inning win.
The Tigers played through a light rain for most of the game. The other two games scheduled for that day were rained out and postponed to Friday.
Chokio-Alberta defeated Hillcrest Lutheran Academy 2-1 to advance to the sub-section championship against Wheaton/ Herman-Norcross. The Spartans lost 11-0, but both teams advance to the Section 6A tournament in Fergus Falls this week.
Wheaton/H-N 11
Morris Area 1
"The game was tighter than the score indicated," said coach Lyle Rambow.
Scoreless after two innings, Dustin Sauter was dueling with the Warriors' pitcher Jay Nelson.
"Our chance to open a nice lead was thwarted by some bad luck," Rambow said.
Mike Siegel was on first with one out when Chris Boettcher doubled off the left field fence. Then Tyler Hansen singled sharply to left. Siegel scored, but as Boettcher raced around third for another run, he slipped and fell in the rain-soaked grass and was nailed between third and home.
The play cost the Tigers a run and an out.
Sauter then singled and Matt Wohlers was intentionally walked to load the bases, but a strike out ended the inning.
In the bottom of the third, the Warriors scored four runs on two hits including a homer by Sam Thielen, a hit batter, and two Tiger errors. One of the errors occurred when a Tiger outfielder slipped down on an easy catch.
Sauter gave up seven runs on the mound, but only one was earned as Morris Area committed four errors.
The Warriors made the Tigers pay with clutch hits to drive in runs. They committed no errors in the wet conditions.
Morris Area had base runners in every inning but the first, but couldn't find the big hits. The Warrior fielders made every out count.
"Our guys improved throughout the season and played hard," said Rambow. "Unfortunately, we made some errors, walked too many, and had some weird bad breaks.
"You won't beat a good team like Wheaton/ Herman-Norcross that way."


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The tech community and others are mourning the death of Aaron Swartz, who committed suicide in his Brooklyn apartment.
Swartz, 26, invented the RSS feed protocol when he was just 14 and went on to help in the creation of the popular social networking site, Reddit.
But last year, Swartz was accused of illegally downloading research documents from the academic service JSTOR, using networks at the Massachusetts Institute of Technology. If he had been convicted, he could have faced up to 35 years in prison.
Now, a petition has been signed by more than 28,000 people on the White House's We The People site. It asks for the removal of U.S. Attorney Carmen Ortiz from office for what they call an overreach in Swartz's case.
Lawrence Lessig, a professor of law and leadership at Harvard Law School, says characterizing Swartz's death as part of the ongoing battle for mental health services isn't a fair characterization.
"I think Aaron was rationally depressed, he was depressed because he recognized that the government was going to pursue this vindictive, completely over-reaching prosecution to the bitter end," he said.
For 18 months, Swartz and his lawyers tried to settle the case outside of court, and originally JSTOR wasn't interested in pressing charges, Lessig said. But prosecutors wanted to make an example of Swartz, and saw parallels between his case and those of people who leaked information to Wikileaks.
"The reality is, every single one of us could be prosecuted by the federal government for something," Lessig said. "What we rely on is good judgment, decency, integrity, balance, proportionality. Those are the principles that a prosecutor has got to hold right in the center of their attention."
Lessig says he know many prosecutors, and many, he says, are the most decent human beings who do what they believe is right.
"But the problem is, when a prosecutor steps out of that legitimate, decent role, we have nothing in our legal system to reign them back," he said.
Whether Swartz's downloads was an act of liberation, or one of theft, Lessig says it depends. This, however, wasn't the first time Swartz got in hot water for making public documents that were access-protected.
Swartz previously made public documents from the PACER federal court document database, Lessig said. Originally he was supposed to be prosecuted for this, but the court overseeing the case determined what he had done was legitimate, not illegal.
Though Lessig doesn't believe copyrighted material should be taken and spread in violation of copyright law, he agrees with Swartz that copyright law needs to be updated and brought into the 21st century.
"I'm not somebody who believes that anybody is entitled to break into a database and take material that is protected by the law, the law, properly conceived, should be enforced," he said.
As for the free culture movement Swartz was part of, it's based on the idea that people should have the ability to build upon and share their culture.
"It doesn't believe copyright should be eliminated, it believes copyright needs to be balanced and updated and he was one of the most important advocates of that movement," Lessig said.


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American mezzo-soprano Lorraine Hunt Lieberson died on July 3 at home in Santa Fe. She was 52.
Widely praised by critics and audiences, her superior musicianship and unassuming personality made her a much loved and respected colleague.
Her interests spanned from Baroque repertoire to the contemporary. In 1997, she appeared in the Santa Fe Opera production of Peter Lieberson's Ashoka's Dream. The two eventually married and she continued to perform his work, as well as sing supporting roles in John Harbison's The Great Gatsby and John Adams's El Niño.
She had cancelled a number of appearances in 2005 and '06, and rumors had circulated concerning her continued ill health following a fight with breast cancer several years ago. She kept the details of her situation private, however, and few were aware that her condition had deteriorated so severely.
National coverage: