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Elizabeth Lawrence is this Thursday Island
Seana R. council soon will 0 Comments
Is might occasionally want to spice things up. These are two different places, even naughty ones.
sitcoms stars Lawrence this Thursday is Island Elizabeth lot
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STD Test on Period.


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I nee a toilet that will fit a 9inch rough in any suggestions?:confused:
Get the latest news, updates and offers
from This Old House to your inbox
Wed, 08/29/2007 - 17:39#1
less than a 10 inch rough in
Wed, 08/29/2007 - 21:25(Reply to #1) #2
You can buy an offset toilet flange which should resolve your problem. You might find these at a big-box, but I get mine via the local plumbing supply house.
(If the install is in a slab you'll still have considerable work ahead of you removing a litttle concrete, installing and closing up again)
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subset: commoncrawl


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"This is a delicious variation on potato salad. I found this recipe on the internet after I had it at an event catered by Backyard Burger. I don't know if this is the exact recipe they use but it is very similar. I serve the crumbled bacon separately so the vegetarians in our family can still enjoy."
Bacon Potato Salad With Ranch and Sour Cream
0 recipe photos
READY IN:40mins
Ingredients Nutrition
- 2267.96 g red potatoes, boiled, sliced
- 236.59 ml Hellmann's mayonnaise
- 236.59 ml sour cream
- 226.79 g bacon, fried, drained, crumbled
- 118.29 ml green onion, sliced
- 2 hard-boiled eggs, diced (optional)
- 1 stalk celery, finely diced
- 226.79 g packet ranch dressing mix
- 63.78 g cansliced black olives (optional)
- crumbled blue cheese (optional)
- Mix potatoes, mayonnaise, sour cream, bacon, green onions, eggs, celery, and dressing mix. Add equal parts of more mayo and sour cream if not moist enough. Put in bowl, chill. Top with bleu cheese and black olives. (They can be optional).
- This recipe also makes a great chicken or turkey salad, substitute the potatoes with the meat, and add some chopped pecans or almonds.


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My husband and I just purchased a home and we are so excited to move in! For some reason we like to do everything all at once around here. i.e. have a baby, move into a new house, start DIY project on said house. It would be nice if we could spread the joy a little but that's ok, keeps life interesting right? With everything going on I knew I had to stay organized and eat healthy! The last thing I want is for my boys to do nothing but watch TV and eat junk all day. It just isn't an option. So I set out to find quick, delicious, and healthy meals I could prepare for them in a hurry.
How To Stay Organized and Eat Healthy
I've been wanting to purchase foods with less preservatives, and added salt and sugar but it has been really hard to get around to doing it for some reason. It dawned on me that no one else was going to do it so I needed to get my act together and I'm so glad that I did. Now on nights when I'm up to my eyeballs in boxes, and my boys are starving, I don't feel guilty giving them Annie's Homegrown Organic Macaroni & Cheese because of this…
- No artificial flavors, synthetic colors or preservatives
- 10g protein per serving
- Produced without antibiotics, synthetic hormones or persistent pesticides
So how do you stay organized and eat healthy when you're beyond busy?
Believe it or not I actually feel more in control right now, with all of this craziness going on, than I have in a long time! I attribute it to planning and making our weekly lunches and dinners on Saturday and getting to bed early. Seems crazy right? But I have noticed a huge difference! Planning our meals and making them ahead of time has helped make us much more successful at eating healthy. We don't have a desire to grab take-out because that would actually take longer than heating up our delicious meals at home. Getting to bed early has given me the extra energy I need to be productive the whole day. Normally I get tired and take a nap with the boys but now I get my "chores" done while they nap. Instead of staying up until 2 A.M!
Have you ever tried an Annie's Homegrown product? Which one are you try next?
You have to try their Shells & White Cheddar Macaroni & Cheese because it really is amazing!


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iShopping.pk brings you the best price for Garmin vivoactive Sunlight-Readable GPS Smartwatch Black with free shipping all over Pakistan.
GPS Smartwatch for the Active Lifestyle
• Ultra-thin GPS smartwatch with a sunlight-readable, high-resolution color touchscreen• Built-in sports apps, including GPS-enabled running, biking and golfing plus swimming7 and activity tracking¹, let you view your stats even when away from your phone• Pairs with your smartphone to gently vibrate and display alerts for incoming calls, texts, emails and calendar items plus notifications from social media and other mobile apps• Customize with free watch face designs, widgets and apps from our Connect IQ™ store• Battery charge lasts through all your activities with up to 3 weeks6 in watch/activity tracking mode or up to 10 hours using GPS
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1.72" x 1.52" x 0.32" (43.8 x 38.5 x 8.0 mm)
Without bands: 18.0 g (0.63 oz); With bands: 38.0 g (1.34 oz)
Water rating
Battery type
Rechargeable lithium battery
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1.13" x 0.80" (28.6 x 20.7 mm)
Display resolution
205 x 148 pixels
Color display
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Up to 3 weeks (10 hours in GPS mode)
High-sensitivity receiver
Clock Features
time, date, calendar, and alarm
GPS Time Sync
Automatic daylight saving time
Alarm clock
Daily Smart Features
Connect IQ™ (downloadable watch faces, data fields, widgets and apps)
Smart notifications
Music controls
Face It™ watch face creator
Find my Phone feature
VIRB® remote
Compatible with Garmin Connect™ Mobile
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Step counter
Move bar (displays on device after a period of inactivity; walk for a couple of minutes to reset it)
Auto goal (learns your activity level and assigns a daily step goal)
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Calories burned
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GPS speed and distance
Customizable data pages
Auto Pause®
Auto scroll
Auto Lap®
Manual lap
Customizable alerts
Vibration alerts
Activity history on watch
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HR zones
HR alerts
HR calories
% HR max
% Harar
Running Features
GPS-based distance, time and pace
Foot pod capable
Yes (optional)
Outdoor Recreation Features
Back to start
Cycling Features
Time/distance alerts (triggers alarm when you reach goal)
Swimming Features
Pool swim metrics (lengths, distance, pace, stroke count/rate, calories)
Pool lap recognition
Stroke efficiency (SWOLF)
Golfing Features
Yardage to F/M/B (distance to front, middle and back of green)
Yardage to layups/doglegs
Measures shot distance (calculates exact yardage for shots from anywhere on course)
Garmin Connect™
Garmin Connect™ compatible (online community where you analyze, categorize and share data)
Automatic sync (automatically transfers data to Garmin Connect)
Pairs with your smartphone to gently vibrate and display alerts for incoming calls, texts, emails and calendar items plus notifications from social media and other mobile apps
On-device data fields/screens: Daily step count, goal countdown, inactivity timer, distance, calories, time of day/date, heart rate*, bike speed*, VIRB® action camera/remote*, music player controls*
Supports heart rate and heart rate zone: Yes (when used with an ANT+ heart rate monitor)
Built-in Sports Apps
vívoactive features built-in sports apps that track your stats — even when away from your phone. Simply tap to choose among GPS-enabled running, golfing and cycling apps plus swimming and activity tracking.
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Whether you're training for a 5K or just for life in general, the running app uses GPS to capture essential running data such as distance and pace. A built-in accelerometer and wrist-based speed and cadence trackers eliminate the need for a separate foot pod when running on an indoor track or treadmill and also when GPS is unavailable. You can set up vibration alerts for heart rate², pace and run/walk intervals. Other training features include Auto Lap® and Auto Pause®. Automatic sync with Garmin Connect™ Mobile gives you a post-run summary, including calories burned and if you scored any personal records, such as your fastest or longest run to date.
Get In Gear
The GPS-enabled cycling app measures time, distance, speed and calories. Compatible with sensors³ such as speed, cadence and heart rate monitors to measure your progress on training rides as well as daily commutes.
Tee Off
Pair with your smartphone4 to download course maps from our golf course database — with more than 38,000 courses worldwide — to play favorite and new courses. The golfing app keeps maps updated and tallies hole number and par. GPS measures layup and dogleg distances, as well as distances to the front, middle and back of the green, to improve usability over the entire hole. Individual stroke play scorecard lets you see if you're under or over par. Measure shot distance at the press of a button. Round timer counts up time on the course, while the built-in accelerometer tracks your steps.
Dive In
Auto Length accelerometer-based technology automatically gauges total and interval distance; laps; calories burned; session average, interval and length pace; and stroke count7, including number of strokes per length and interval and session averages. Let the swimming app calculate your efficiency with swolf, which adds the time and the number of strokes it takes to swim a pool length. View your time and interval distance at a glance. Interval Count lets you seamlessly pause and then resume your workout.
The thin profile cuts through the water, and its 5 ATM water rating means you can swim, sweat and shower with it on, worry-free.
Step It Up
vívoactive follows your progress even when you're not engaged in a specific activity. The activity tracking app tracks your steps to learn your current activity level, then assigns an attainable daily step count goal. As you meet your milestones, the device will adjust your goal for the next day. The red move bar appears on the display after 1 hour of inactivity and builds when you've been sitting too long. Just walk for a couple of minutes to reset the move bar. Set the sleep mode when you go to bed and vívoactive will monitor your rest. It also tracks your calories burned.
Stay Connected
vívoactive gently vibrates to alert you to all the same notifications you receive on your compatible Bluetooth-enabled device5. Receive caller ID and missed call information for incoming calls. Text messages include sender information and message. Email messages come with sender and subject. vívoactive also displays notifications from Facebook®, Twitter® and other social media platforms, as well as other mobile apps, including weather alerts and temperature. Simply touch and swipe the screen to read more, or clear the screen with the press of a button.
Advanced Smartwatch Features
vívoactive lets you control the tunes on your music player, locate your phone and let your friends and family track your activities in real time with Live Track. You can also remotely control your VIRB® Elite action camera.
Custom Apps and Watch Faces
Personalize vívoactive with free downloads from our Connect IQ store. You can customize your watch face, add data fields and get apps and widgets that provide information at a glance.
Challenge Yourself
Stay up to date on your progress for each of the built-in activities with automatic syncs with Garmin Connect Mobile. You can earn virtual badges for extra motivation, join online challenges and connect, compete and compare with friends and family around the world. View your history and stats right on the smartwatch.
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vívoactive comes in basic black or bright white to coordinate with your philosophy of style. Swap out additional bands to show your true colors, or dress it up with a leather option in black or white.
Wear Your Personality on Your Sleeve
Download the free Garmin Face It™ watch face app to customize your device with any photo from your smartphone. Just download the app, choose an image from your photo library, adjust the style for time and set it as your watch's face. Whether it's a scenic view from your last hike or a picture of your new puppy, with Garmin you can add personality to your watch that's uniquely you.
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- Doctors
- Louisiana
- New Orleans
- Gynecologists
- Dr. Rebecca U. Perret
You may also like
Ratings for Dr. Rebecca U. Perret
I chose Dr. Perret because I wanted a natural delivery with an accomplished physician just in case anything went wrong. My experience during prenatal care was okay. Dr. Perret herself was always very warm and engaged. She was very thorough in her explanations, shared a lot of information and asked if I had questions. The downside was that she always ran very late. I typically waited 90 minutes to see her. Her nurse is not detail-oriented and frequently got information wrong or it was incomplete. Dr. Perret was on-call the night I delivered but didn't answer her phone when the hospital called her. I waited for over an hour fully dilated for the nurses to track down another doctor to deliver my baby. Finally another doctor from her practice arrived and delivered my baby. When that doctor arrived, she made a point of saying that she didn't understand why any woman would deliver without drugs. She said that she had two boys and she got an epidural ASAP. I have no idea why she felt that was useful information to share with a woman laboring without drugs (and who was handling it without too much trouble). I never heard from Dr. Perret while I was in the hospital after delivery, and never received a follow-up call to see how the delivery went, or anything. I was pretty shocked at how extremely uncommunicative she was around the delivery. The labor & delivery nurses were as well and commented that this was not typical for her. I didn't see/hear from her until a follow-up appointment after delivery. She never referenced where she was the night I delivered, why she didn't answer her phone or why she didn't call to follow up.
I love my doctor, but her staff is very frustrating and difficult. It seems they have made it their jobs to keep you from seeing or speaking with the doctor for as long as possible.
love love love this doctor! she is so sweet and amazing!
I LOVE Dr. Perret! She was very supportive of my decision to have a natural birth. She was not on call when I went into labor, but came to the hospital anyway and was very present during my labor. She helped me work through contractions and made the whole experience much more than I could have imagined. The one negative to her would be the wait time at her office. There were several times during my pregnancy when I waited well over an hour to even go back into the exam room, where I would usually wait another 30-45 minutes to see the doctor. Her nurse at the time was very short with patients and never showed much compassion. Dr. Perret, is wonderful, her office...not so much.
Dr. Perret is the best doctor. When she walked into the delivery room, I was so relieved, because I trust her with my life. She is absolutely the best OBGYN. I recently moved, and I know it will be a challenge to find a doctor as great as her. I am not sure if someone can be so luckly to find a second amazing doctor. I would recommend her to anyone and everyone.
I love Dr. Perret. She's down to earth, very helpful, and very easy to understand. Her staff is another issue. I'm lucky if I wait less than an hour and a half before seeing her and when I've brought it up with her staff they've simply said, "You're not the only patient we have" very rudely and matter-of-factly. I'm honestly considering switching simply because of this. No pregnant woman should have to wait that long at EVERY VISIT (and I'm 20 weeks so it's happened more than once). I'm only conflicted because I've had bad luck with other OBs before.
She is absolutely wonderful; she takes your concerns seriously and would never discount any of your concerns, problems, fears etc. At the same time, she tells you things as they are and will tell you if your concerns etc. are well-founded or not. She has a wonderful mixture of empathy, warmth, humor and, for lack of a better word, "down-to-earth-ness". My first pregnancy and delivery were child's play with her at my and my husband's side. I send all my friends to her.
I feel so lucky to find Dr. Perret as my OB. She is enthusiast, well-mannered, and can always make me feel comfortable. I would recommend her to my friends.
I love Dr. Perret, she has been my doctor for several years now. She is very caring and takes a personal interest in her patients. She has been with me through a miscarriage and the delivery of my twins.
Dr. Perret was the best doctor I've ever had. She has a gift for making people comfortable and is a totally non judgemental person. I really can't say enough about her! I'm so lucky a friend reccomended her to me. She's the best!
Dr. Rebecca U. Perret's Credentials
Facility Affiliations
Accepting New Patients
- Louisiana State University School Of Medicine In New Orleans (Grad. 1998)
Other Specialties
Areas of Expertise
Awards & Recognitions
Publications & Research
Insurance accepted by this Doctor
- Coventry Health Care
- Delta Health System
Top Hospitals in New Orleans, LA
Top Urgent Care Centers in New Orleans, LA
Find a Doctor by Specialty
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Top Gynecologists (OBGYN)
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These 9" long cast urethane skulls not only glow in the dark after exposure to sunlight, they also light up! The sconce hangs using a metal loop or can be displayed on a table top.
Purple skulls will come with amber LED. Black will come with teal LED.
Each skull comes with a small LED tealight that can be set to flicker like a candle, flash or have a steady glow. It is turned on with a button at the top of the tealight itself and is then snugly placed into the crystal. You can also use a remote control for these lights if you want more convenience ( buy it here) . These light up with a bright teal color. The batteries will be shipped outside of the light to preserve the battery life.
Please be sure to screw in the LED light base tightly before turning it on. This will allow the battery to work correctly.
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Resources and Best Practices
Searching for your next leadership opportunity in healthcare, education, academic medicine, or the non-profit space? These are our areas of specialty, and we understand executive career challenges and issues inside and out. WK Advisors' experts have written or weighed in on today's most important career issues in the articles below.
Prepping Job Candidates for Interviews Increases the Odds for Success
Diane Nicholas, consultant, shares best practices with Roy Maurer of SHRM.org about how recruiters can work with job candidates to prepare them for the rigors of the interview process.
8 Ways Candidates E-Sabotage Themselves
The Internet is a powerful job search and career tool, but it must be used carefully and wisely. In Training Magazine, consultant Diane Nicholas offers cautions for executives regarding online career activity.
Find Solid Footing in a Shifting Career Landscape
Practice Leader David Boggs provides fundamental career advice for executives in industries undergoing significant change.
Starting a Job Search: 5 Questions to Ask Yourself First
Consultant Diane Nicholas discusses five critical questions to help you narrow down what you really want in your next job.
Write an Awesome Resume: An Executive Recruiter Gives Insider Tips
April Allen, an Associate at WK Advisors, offers her insider tips on Brazen Careerist for revising a resume for the digital age.
How to Land the Job You Really Want—Now
In The Fiscal Times, WKA Practice Leader David Boggs discusses broadening your horizons to find the career opportunity of choice.
Should You Tell Your Boss You Are Job Searching?
From About.com's Job Searching Expert, Alison Doyle, comes advice on when — or if — you should tell your boss about a job search or interview. WK Advisors Practice Leader David Boggs weighs in on the decision between transparency versus confidentiality.
Six Tips for Unemployed Executives
On CNBC.com, WK Advisors Practice Leader David Boggs offers up career advice for out-of-work executives.
7 Resume Myths That Won't Get Your Hired
On GlassDoor.com, WK Advisors' David Boggs joins other experts in refuting some of candidates' commonly held resume beliefs.


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no subjectDate: 2012-09-28 08:24 pm (UTC)
From: madlybewildered.livejournal.com
I'm with you on that. One of the things I hate most about my brilliant plans is not being able to gloat about my brilliant plans because the best plans are the ones that no one notices being enacted. Sigh.
no subjectDate: 2012-09-28 09:00 pm (UTC)
From: y3llowdaisi3s.livejournal.com
I monologue a lot. I run my own business with my best friend and we monologue a lot when we are planning ahead. You have no idea how much more fun it is to talk about the Quarterly numbers in the evil villain voice and go on and on about what you plan to do in the next month. Totally better than a typical boring business meeting, let me tell you.
What do you want to monologue about?
What do you want to monologue about?
no subjectDate: 2012-09-28 10:32 pm (UTC)
From: unpossibly.livejournal.com
I monologue because if I don't, I can go through an entire day without speaking. Unfortunately since I rarely have an audience, I monologue to myself. (If I monologue in a forest, and no one is around, does it still count?)
no subjectDate: 2012-09-29 05:41 pm (UTC)
From: scatteredintime.livejournal.com
You and me both.
And how dare you link to tvtropes, I'll never get around to the Hogsmeade Spreadsheet.
And how dare you link to tvtropes, I'll never get around to the Hogsmeade Spreadsheet.
Edited Date: 2012-09-29 05:41 pm (UTC)
no subjectDate: 2012-09-30 07:15 pm (UTC)
From: http://users.livejournal.com/unnecessary_/
Please explain why! I feel like my writing would benefit from your explanation!


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Phone.com keeps on adding on features and hits for the SMB crowd. The company has added Mobile Office for BlackBerry to its services.
Customers can now listen/manage voice mail, view received faxes from a phone, run Call Logs on a call extension, block calls, quickly connect to a conference bridge, and make calls from a Phone.com virtual office number with the number popping up on the inbound caller ID.
Best of all, the BlackBerry app is free to download and available with all Phone.com plans.


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Verse 5. Because they regard not the works of the Lord, nor the operation of his hands. God works in creation -- nature teems with proofs of his wisdom and goodness, yet purblind atheists refuse to see him: he works in providence, ruling and overruling, and his hand is very manifest in human history, yet the infidel will not discern him: he works in grace -- remarkable conversions are still met with on all hands, yet the ungodly refuse to see the operations of the Lord. Where angels wonder, carnal men despise. God condescends to teach, and man refuses to learn. He shall destroy them: he will make them "behold, and wonder, and perish." If they would not see the hand of judgment upon others, they shall feel it upon themselves. Both soul and body shall be overwhelmed with utter destruction for ever and ever. And not build them up. God's cure is positive and negative; his sword has two edges, and cuts right and left. Their heritage of evil shall prevent the ungodly receiving any good; the ephah shall be too full of wrath to contain a grain of hope. They have become like old, rotten, decayed houses of timber, useless to the owner, and harbouring all manner of evil, and, therefore, the Great Builder will demolish them utterly. Incorrigible offenders may expect speedy destruction: they who will not mend, shall be thrown away as worthless. Let us be very attentive to all the lessons of God's word and work, lest being found disobedient to the divine will, we be made to suffer the divine wrath.
Verse 4-5. See Psalms on "Psalms 28:4" for further information. In these verses, as indeed in most of the imprecatory passages, the imperative and the future are used promiscuously: Give them -- render them -- he shall destroy them. If therefore, the verbs, in all such passages, were uniformly rendered in the "future," every objection against the Scripture imprecations would vanish at once, and they would appear clearly to be what they are, namely, prophecies of the divine judgments, which have been since executed against the Jews, and which will be executed against all the enemies of Jehovah, and his Christ; whom neither the "works" of creation, nor those of redemption, can lead to repentance. George Horne.
Verse 5. Culpable negligence constantly persisted in, losing much blessing, and involving terrible condemnation.


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Finished size of the handbag is 6-1/2"x4"EAN: 082676206139
I have worked many and they are really nice! product absolutely recommended!
purchased and set aside for the moment I have to find a suitable yarn (for my taste) fills exposed well and the result satisfies me as an idea but really cute! especially for girls who can create their own little bag or pochette! low price and excellent workmanship


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Friday, 6 January 2017
Brass Band Buizingen is delighted to invite you to their 24th Valentine Concert on Saturday Feb 11. For this festive occasion, MD Luc Vertommen has selected a lovely programme and a special soloist: principal trumpet of the London Symphony Orchestra Philip Cobb.
Monday, 28 November 2016
For the second year in a row, Brassband Buizingen was crowned champion of Belgium. They will represent Belgium at the 2017 European Brass Band Championship in Ostend.
Monday, 10 October 2016
Steven Mead and Brass Band Buizingen cooperate again after eleven years. Steven Mead, renowned for his lyrical phrasing and extraordinary and virtuoso technique is accompanied by 'a brass band at the top of its form and led by a fantastic MD who arranged many of the pieces for brass band' (quote: Steven Mead)
Thursday, 7 July 2016
It's now 10 years since Steven Mead recorded his award-winning album 'Euphonium Virtuoso' with Brass Band Buizingen and conductor Luc Vertommen. The band have gone from strength to strength and recently achieved a magnificent fourth-place at the recent European Brass Band Championships in Lille.
Friday, 15 April 2016
Tuesday, 23 February 2016
Brass Band Buizingen's new principal cornet Dobbelaere Lieven made his debut with the Belgian champion in a special Valentine's Concert last weekend.
Monday, 4 January 2016
Brass Band Buizingen is delighted to invite you to their 23rd Valentine Concert on Saturday Feb 13. For this festive occasion, MD Luc Vertommen has selected a lovely programme and a special soloist: trombone player Jörgen van Rijen.
Monday, 14 December 2015
After Brassband Buizingen regained the Belgian National title, Harmen Vanhoorne decided to leave the band when they are at a high.
Tuesday, 3 November 2015
On 17 (Meise) and 31 (Merchtem) October Brass Band Buizingen conducted by Luc Vertommen cooperated with more than 100 singers from the united choirs of the villages of Meise and Merchtem to perform (for the 3rd and 4th time ever!) the Baron d'Hoogvorst cantate.
Wednesday, 6 May 2015
We would like to present to you our crowd funding project "The complete oeuvre for wind band by August De Boeck (1865-1937)".


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Station: Earth's Interior
In this earth science worksheet, students cut out each die pattern and the signs for each station. Then they assemble the dice by folding along lines and taping the edges together. Students also follow the written instructions for traveling through the rock cycle.
12 Views 96 Downloads
Resource Details
Shake it up with Seismographs!
Shake things up in your STEM or earth science classroom when you have small groups construct their own seismographs. A reading assignment on the history of seismographs, the Richter scale, and current technology sets the stage for the...
3rd - 12th Science CCSS: Adaptable
Middle School Sampler: Science
Focus on inquiry-based learning in your science class with a series of activities designed for middle schoolers. A helpful packet samples four different texts, which include activities about predator-prey relationships, Earth's axis and...
6th - 8th Science CCSS: Designed
Earthquake Education Curriculum
In case of earthquake, go to a safe place before posting on social media. The unit includes 12 lessons, most with hands-on activities or experiments. The first six lessons cover the physical process of earthquakes and volcanoes. Lessons...
6th - 12th Science CCSS: Adaptable
Regents High School Examination: Physical Setting Earth Science 2006
Test your class on earth science with this extensive resource. This test, created by The University of the State of New York Regents, is made up of 50 multiple choice questions and 32 short answer questions that cover the branches of...
9th - 12th Science
Space Station Research Explorer
Take a trip into outer space from the safety of your classroom. A great addition to the digital library of any science teacher, this reference offers a behind-the-scenes look at the research going at the International Space Station.
4th - Higher Ed Science CCSS: Adaptable
Start Your Free Trial
Save time and discover engaging curriculum for your classroom. Reviewed and rated by trusted, credentialed teachers.Try It Free


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National Geographic
Making Ethical Art From Dead Animals
National Geographic
Kimberly Witham's roadkill art isn't like the traditional stuffed animal trophies the word "taxidermy" usually implies. Rather than killing animals just to hang their heads on her wall, Witham recovers the roadside remains of already-dead critters …
Source: animals – Google News
Sharing information is key – Animals Business News UK. Social Commerce and Social Media Management playing a big part. Suaju Ltd


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Letona Motorcycle StorageClose Map
Easily compare Prices, Discounts, Photos and Reviews. Letona vehicle storage facilities.
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Papis Takes Lexus Sixth Pole In Seven Rolex Series Races LEXINGTON, Ohio (Aug. 6, 2004) - With co-driver Scott Pruett absent while qualifying for Sunday's Brickyard 400, teammate Max Papis didn't skip a beat as he gave Lexus and the CompUSA ...
Papis Takes Lexus Sixth Pole In Seven Rolex Series Races
LEXINGTON, Ohio (Aug. 6, 2004) - With co-driver Scott Pruett absent while qualifying for Sunday's Brickyard 400, teammate Max Papis didn't skip a beat as he gave Lexus and the CompUSA Ganassi Racing team its sixth pole in seven Grand American Rolex Series races this season in preparation for Saturday's EMCO Gears Mid-Ohio Road Racing Classic.
The pole marks the second time Papis has earned the top spot in qualifying this year as he set a new track record in the process. Papis and Pruett own an 11-point lead in the championship race heading into this weekend's action.
"We are very, very pleased with the balance in our race car," Papis said. "We worked very hard on race setup, but we didn't work that hard with getting the best out of the car. We are pleased with how well we did in qualifying. We are pleased with both cars. With the (team's) No. 02 (car), Jimmy (Morales) is making a lot of progress. As usual, I think it's going to be a very close battle."
Among those making up that close battle will be local favorites, Michael Shank Racing (MSR). Competing an hour from the team's shop in Columbus, Oswaldo Negri registered the fourth-fastest lap to qualify on the outside of Row 2. The #6 MSR Lexus also was second in Friday's first practice session.
Rounding out the top five was the #02 CompUSA Lexus of Jimmy Morales and Luis Diaz - the Ganassi team car to the Papis/Pruett entry. The fifth-place qualifying effort marked the fifth time this season that the #02 has qualified in the top five.
Another other Ohio-based Lexus team, Doran-Lista Racing, registered an eighth today to make it four Lexus-powered entries in the first eight cars. Veteran road racing champion Didier Theys qualified the car and will team with 22-year-old Swiss driver Benjamin Leuenberger. Both regular team driver Fredy Lienhard and the team's third driver, Jan Magnussen, had other commitments, but Magnussen will return to drive next weekend at Watkins Glen.
The EMCO Gears Mid-Ohio Road Racing Classic is tentatively scheduled for a 5:30 p.m. start on Saturday, with SPEED's tape-delayed coverage beginning while the event is still in progress at 8:00 p.m.


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Nominations - August Dramatic Post and Blogsite
These are the nominations so far for Best Dramatic Post and Blogsite - there is still time to nominate up to 11:59 PM, August 31st!
I apologize for not getting these out to you sooner. But I'm really no good at trying to put links in, and although some of you generously provided them for me (and I owe you a debt of undying gratitude), others did not and I am not the Net-savviest creature on the planet. But I am learning!!!
Without further ado - thus far, the nominees (listed in no particular order) are:
Best Dramatic (Serious) Post:
Mossy Stone - - Withering Heights
Danikabur - I've been Mulling Over Something
Danikabur- Continuation
Callie -A Whole Lotta Trouble: Looking into the Past
Lowk - life or something like it: Scary Movies
Lab Boy - Dad is 51 today
David Hagstar -Enter The Wardrobe: Bad Day, Good Medicine
Best Dramatic Blogsite:
G.D. - Lions and Tigers and Maggots, Oh My...
Lawbrat - Lawbrat.com
81vaginas - 81 Vaginas, A Pillow Blog
Paul -A Spiritual Diablog
Didn't see your favorite dramatic post or blogsite here? Nominate it, in the comments. Nominations end August 31st at 11:59 PM, voting starts September 1st and end September 5th at 11:59 PM!!
(Moderator's Note - I apologize for misnaming G.D.'s post in the nomination. I have corrected the error and appreciate your indulgence.)


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AN T-OBAN - The little bay in Gaelic
Started Jun 5, 2014 | Photos thread
Manzur Fahim • Senior Member • Posts: 1,486
AN T-OBAN - The little bay in Gaelic
Jun 5, 2014
Hello everyone,
Hope you are well.
I wanted to share my trip to Oban - little Scottish town between Fort William and Helensburgh.
I was only there for a day, so just walked around took some of photo of the town and some photos of Isle of Mull from Isle of Kerrera.
All photos taken with Nikon D800E + Tamron 24-70mm f/2.8 VC
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Manzur Fahim's gear list:Manzur Fahim's gear list
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Comment & critique:
Please provide me constructive critique and criticism.
Nikon D800E Tamron SP 24-70mm F2.8 Di VC USD
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Mothers' Day Gift Ideas
May 12, 2017
Filed under Facts-Jokes-Riddles, Lifestyle
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Mothers' Day is this Sunday and if you haven't gotten her anything or even have an idea as to what to get your precious mother, here are some ideas:
Okay, yeah, I know it's not a big thing but it's something. You can go to any store like Smart & Final, Sam's Club, or Vons. It'll show her that you thought about her.
You can give a letter telling her how thankful you are and how much you love her. Personally, I do that when I don't have money, which is mostly every year.
Take Her Out To Eat:
If you can, take your mom out to lunch or something and have a nice time with her. You can go to Olive Garden or Lazy Dog. Or just plain out ask her where she wants to go and eat there.
Make Her Something:
Even if it doesn't mean that much to you, it could mean the world to her. It would show her you care. You put thought into it and she'll appreciate it. It could simply be a drawing or a little post it note saying "I love you", I'm almost 100% sure she'll be more than happy.
Hopefully those helped those who still don't know what to get their mothers on Mothers' Day. Happy Mothers' Day!


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Robienie biżuterii zrób to sam
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DIY Tutorial: How To Start Making Jewelry / Jewelry Making Basics - Visual Guide to Jewelry Supplies
How to make classic hoop earrings that will never go out of style: FREE jewelry making tutorial!
'17 Easy and Creative DIY Jewelry Making Projects Perfect for Gift Giving...!' (via Ideal Me)
20 DIY Earring Projects | My Girlish Whims... there are plenty of other amazing ladies out there in blog land who share great jewelry making tutorials as well. So today I've rounded up 20 DIY earrings projects from myself and some other crafty ladies. Enjoy!.
How to: Knots used for fishing lures can also be helpful in jewelry making ✿...
Are you new to jewelry making? These top 10 tips will help guide you in the right direction! (and if you're a seasoned pro, you might learn something too)
Jewelry Making Basics - Visual Guide to Basic Jewelry Tools
Jewelry Making! DIY Braided Bracelets | http://diyready.com/4-wire-jewelry-making-techniques-diy-bracelets/
Necklace lengths. Good to know!- Great for helping DIY jewelry making.- More


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Basil Pork Chops
4 Lean center-cut pork chops (1 1/4 lbs.)
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1 ts Dried basil
1/2 ts Salt
1 ts Coarsely ground black pepper
Trim and discard excess fat from chops. In a large frying pan, brown chops without added fat. When chops are browned, add other ingredients. Cover tightly and simmer 40 minutes or until tender. Turn meat occasionally and add a few tablespoons of water if necessary to prevent burning.
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eReplacements LMP-M130 - Projector lamp - 132 Watt - 900 hour(s)Item#: 8382621 | Model#: LMP-M130
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Projector Lamp for Sony Product Details
Product Type: Projector lamp
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Designed For: Sony VPD-MX10
Manufactured by: eReplacements
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use jotty's or kapersky, lol, we have so much of these, but gj... not rlly...
Hi, Tortaleni here.. here is a new release of my CA TOOL it has
** CBL Checker
** CA Remover
** IN Game Spammer
Now, i didnt take any pics. i took a vid, its easier!! so view it at bottom
Virus Scan
Virus Scan II
Video [YOUTUBE]<object width="640" height="505"><param name="movie" value="https://www.youtube.com/v/417r50WgrXk&hl=en_US&fs=1&color1=0xcc2550&color2=0 xe87a9f&hd=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="https://www.youtube.com/v/417r50WgrXk&hl=en_US&fs=1&color1=0xcc2550&color2=0 xe87a9f&hd=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="640" height="505"></embed></object>[/YOUTUBE]
Last edited by Liz; 03-06-2010 at 09:58 PM. Reason: Adding Virus Scans.
use jotty's or kapersky, lol, we have so much of these, but gj... not rlly...
Jotti's malware scan, novirusthanks, etc
Looks nice.... But need a VS
Added and fixed virus scans.. and approved...
Click Here to Become a MPGH Premium Member by Donating $25 or More.
If anyone claims to be me via MSN/Skype/Email or any other source outside of MPGH private or visitor messages, IT'S NOT ME!
They are trying to trick or scam you. Report them immediately and PM me here for verification.
"Dont confuse my personality with my attitude. My personality is who I am. My attitude depends on who you are." Frank OceanModerator: 5/2009-10/2009 | GMod: 10/2009-10/2010 | Staff Administrator: 10/2010-Present
Stupid/Pointless Private messages = SPAM, SPAM = BAN.
Just found out that CBL is updating to 2.0 ... Can't even check if Im dirty or not
v2.3 will be released sometime soon with :
** Spammer (Newer)
** Tapper (same)
** CBL Checker ( Same )
** CA Remover.
New Thins im going to add are :
** Submit Bug Report System
** Remove Recoil ( All game hack) (If I Can be bothered)
and More if i can find more things to add..
Please give me some idea of what to add to it.
P.S I cannot Code Hacks or anything like that so dont put any sort of hack on the list!
But seriously you should give credit to the people who's tutorials you read and copied code from.
ummm. i gave credits to Ugleh, for his TuT ( CA Remover, Tapper ) but the rest is all mine.
Last edited by D3Dh0oker; 03-07-2010 at 08:43 AM. Reason: Spelling mistakes :O
Well, in all fairness not much code actually goes into these tools. I mean I can code spammers in 3 minutes.


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The HU Chant
Everything that life serves up for us in any given moment is an opportunity to learn, grow and remember more about who we are and the lessons we're here to learn. As we progress through life spirit works with those that seek more understanding and truth. As a result, many are motivated to not just seek but to express it in their lives.
Just like we exercise physically, there are spiritual exercises we can do and spiritual disciplines we can practice to bring spirit into our lives for our growth and the blessings of others. In my personal life, I've searched for effective ways I can connect with spirit. There are many paths, beliefs and practices. However, I've found that the more simple, clean and pure we can keep it the better. The more encumbered it is with the 'opinions' of man, the easier it is to only practice things and try to understand things on an intellectual basis. Consequently, we defer and put off the personal adventure of experiencing the 'spirit' in our own lives and experiencing the growth we most need to learn the spiritual lessons we came here to do.
I've personally experienced 'spirit' to be a form of energy. The creative energy we've come to 'label' as God. However, in the end it's energy. I take seriously the words of Christ when he said, "ye are Gods". For that he was put to death. Even today, espousing that truth will get one 'sideways' not only with society, friends and even family, but with religious authority. To make the claim that we are 'Gods' is to go against all the reasons for which men seek to 'lord' over others and make them 'less than' who we really are...soul beings, children if you will, of the Father (spirit). The Master Teacher, Christ (along with many other masters) exemplified the way we must live to 'invite' spirit into our lives...a process that catalyzes our spiritual growth and 'brings all things to our remembrance'.
Once a person has 'seen God', there is no reason to share it with anyone else...all that matters to that person is to desire to do the 'will of God' and become a 'co-worker or co-creator' with God. Throughout scripture and the ancient texts, this principle is plainly taught. At an appointed time we seek to 'co-create' with God and be instruments in his hands for 'building up the kingdom' here on the earth.
The HU
Throughout the ages, followers of many spiritual traditions have used prayer, the singing of holy words, and meditation to bring themselves closer to God. In the same way, those who have discovered HU, an ancient name for God, sing it for their spiritual upliftment.
Regardless of your beliefs or religion, you can sing HU to become happier and more secure in God's love. Singing the HU connects everyone with the heart of God. This Light and Sound is literally the Holy Spirit. Direct experience with these twin aspects of God opens the deep spiritual potential within each of us.
The Light and Sound purify, uplift, and direct us on our journey home to God. Singing HU draws us closer in our state of consciousness to the Divine Being. This is its purpose. It is for those who desire spiritual love, freedom, wisdom, and truth.
Upliftment throughout Your Day In The Golden Heart, Mahanta Transcripts, Book 4, Harold Klemp explains how HU can help you see your life from this higher viewpoint.
"As you chant the name of God, with love, for twenty minutes a day, the bindings and bands that constrain Soul will begin to unwind. Not all at once, but very slowly, at a rate you can understand and accept. As these bindings are released, Soul rises in spiritual freedom.
"As Soul, you are like a balloon that rises above the ground. The higher you go, the farther you can see. And the farther you can see, the better you can plan your life."
HU is woven into the language of life. It is the Sound of all sounds. It is the wind in the leaves, falling rain, thunder of jets, singing of birds, the awful rumble of a tornado. . . . Its sound is heard in laughter, weeping, the din of city traffic, ocean waves, and the quiet rippling of a mountain stream. And yet, the word HU is not God. It is a word people anywhere can use to address the Originator of Life.
The Light and Sound of God together are the Voice of God. In scriptures we know It as the Word or Logos. In other words, it's God speaking from beyond creation to Its creation.
The Holy Spirit Is Energy, Light or Frequency The Light and Sound are the twin aspects of the Holy Spirit, the divine life current that flows from God. This current sustains all life. Light and Sound have long been a part of religious rituals throughout the world. Chants and hymns are among the most well-known examples of sound in the Christian tradition. The singing of cantors plays an important role in Judaism. Om, sung in some Eastern religions, is often used in meditation.
Likewise, the Light of God is symbolically represented in most religions. Stained glass, precious stones, and skylights are common in religious structures. Native American celebrations, as well as those of certain other religions, use fire and drums, as well as chanting, all examples of light and sound. Each ceremony represents the effort of people to speak to God in their own way.
The Light of God has appeared to many saints and mystics. Saul of Tarsus had a dramatic encounter with the Light of God on the road to Damascus. Moses saw It in the burning bush. Throughout history light has been depicted around images of holy people and in illustrations of angels. Today, many refer to this light in popular books about near-death experiences.
Your Experience of the Light and Sound of God Is Personal To YouWhile others might be able to help you to recognize the Light of God. Your experience is personal to you. Sometimes It comes as an inner scene or vision. It can be a flash of blue or white light. It appears in many ways. The Sound of God was the rushing wind that visited the disciples during Pentecost. Some hear It inwardly. It can be heard as the sound of rushing water; the single note of a flute; the music of violins, woodwinds, or bagpipes; or even the buzzing of bees.
For people who experience these twin pillars of God,often through practicing the HU chant and other spiritual exercises, life becomes fresh and new again. As we unfold spiritually, we learn to express the love of God through service to others.
Experience HU
To do the exercise, first get comfortable. Close your eyes and take a few deep breaths. Next, gently put your attention on your inner visual screen, where daydreams and images come to you.
With your eyes closed, sing HU (pronounced like the word hue) as a song of love. You may notice a feeling of peace, warmth, and comfort. You may also recognize the presence of the Divine through greater spiritual insights into your daily life. There are many ways to experience the love of God.
When you sing HU and sit in quiet contemplation, you might also perceive the inner Light and Sound. The Light may appear as brightness or colors on your inner visual screen. The Sound may be musical or the sounds of nature, such as the wind or the ocean. However you experience the presence of God, it is bringing you a broader understanding of the life you lead.


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Fig. 33.1
A famous example of neutrophil chemotaxis and phagocytosis. The neutrophil is seen migrating toward a bacterium as it secretes some kind of unknown chemoattractant (e.g., N-formyl peptides like fMLP). The neutrophil polarizes its cytoskeleton and extends lamellepodia toward an increasing concentration of proteins that guide the cell toward the bacterium. Note the ability of the neutrophil to rapidly change direction and polarization in response to movement of the bacterium, as well as its ability to navigate around red blood cells. In its final fate, the bacterium is engulfed by the neutrophil and killed during phagocytosis. (The above images were taken from a 16 mm video recorded by David Rogers of Vanderbilt University in the 1950s. Details can be found at www.biochemweb.org/neutrophil.shtml)
Neutrophil chemotaxis can be broken down into three distinct but overlapping stages: chemosensing, polarization, and locomotion (reviewed in [44, 45]). When a neutrophil senses a concentration gradient of external secreted proteins known as chemokines, the cell polarizes its cytoskeleton and engages in sustained directional migration toward the attractant molecules (i.e., chemoattractant). We will provide a brief overview of the biology of chemotaxis for neutrophils and refer the reader to the aforementioned reviews for a more in-depth treatment of the subject.
126.96.36.199 Chemosensing
Neutrophils stay in an inactive and immobile state until they are activated by external cues. When a chemoattractant is present, neutrophils extend and retract lamellipodia—protrusions of the cell membrane comprised of actin filaments—to dynamically form pseudopods for about 60s intervals. If a uniform concentration of chemoattractant is present, neutrophils migrate more or less randomly until the cell senses a gradient of chemoattractant and begins to migrate directionally. Neutrophils sense chemoattractants using transmembrane chemoattractant receptors (e.g., fMLP-R and C5a-R) that become occupied by extracellular chemokines, and these receptors activate heterotrimeric G-proteins that transduce the extracellular signals and initiate an intracellular activation cascade. The chemoattractant receptors are relatively uniformly distributed on the cell membrane, and the local and temporary nature of the pseudopod formation allows neutrophils to rapidly change the extension of pseudopodia in reaction to location changes of the source of chemoattractant. Once the cell becomes polarized, neutrophils maintain the original leading edge of the cell and turns toward the new source of chemoattractant rather than forming new pseudopodia.
22.214.171.124 Polarization and Locomotion
Neutrophils can become polarized in a uniform concentration of chemoattractant; however, polarization is much more likely when a concentration gradient exists, resulting in persistent migration toward the source of chemoattractant (chemotaxis). When there is a difference as little as 1–2 % in the number of receptors on opposite ends of the cell occupied by chemoattractants, the neutrophil polarizes its cytoskeleton by increasing the number of advancing pseudopodia on the anterior portion of the cell and enriching retracting uropods with myosin in the posterior portion of the cell. Once activated and polarized, neutrophils have the ability to persistently migrate toward a site of inflammation that is sending inflammatory signals (chemokines) or chase down pathogens such as bacteria to phagocytose and clear the pathogen. The persistent migration of neutrophils is determined by a variety of factors such as type of chemoattractant, steepness, and profile of the concentration gradient and other biophysical and biochemical factors in the neutrophils' extracellular microenvironment.
33.2.2 Chemotaxis in Disease
In the introduction, we described chemotaxis as central to the pathophysiology of many diseases. Indeed, chemotaxis is relevant to the spread of metastatic cancer [36], wound healing [33, 46, 47], the inflammatory response by the innate immune system [48, 49], rheumatoid arthritis [50], chronic obstructive pulmonary disorder (COPD [51]), and asthma [52–54]. The role of chemotaxis in the pathogenesis of these and many other diseases has been intensely studied over the last 50 years. Researchers have made progress in identifying chemotactic cell types central to the pathogenesis of a disease, as well as the identification of cytokines orchestrating cell mobility.
For example, the dissemination of metastatic cancer cells from a primary tumor site throughout the body is central to the pathophysiology of the disease. If a primary tumor is detected early, treatment regimens and prognosis are greatly improved compared to late-stage detection [55]. In other words, the cancer becomes much harder to treat once the cells have migrated away from the primary tumor site, entered the bloodstream (after angiogenesis has been initiated), and extravasated from the blood stream to form a secondary tumor. If the cancer cells have not left the primary tumor site, the cancer can often be surgically removed or treated with local irradiation. Although directional migration is not necessary for this process, there is evidence that metastases occur more efficiently when the cells migrate directionally [56]. Researchers have also shown that the chemotaxis of tumor cells toward macrophages is important for intravasation into blood vessel and for invasion into the tissue of a secondary site [57]. Minimizing or disrupting the ability of cancer cells to undergo chemotaxis may lead to a reduction in the metastasic potential of cancer cells, resulting in improved prognostic outcomes for cancer patients.
In asthma, the dysregulation of inflammatory response contributes to the pathogenesis of the syndrome. Asthma is a condition characterized by chronic inflammation of the lungs that ultimately leads to obstruction of airflow, resulting in clinical symptoms including wheezing, coughing, and shortness of breath. The condition has far-reaching impact and affects more than 300 million people worldwide [58]. Furthermore, asthma prevalence has increased significantly over the last 30 years in many regions, with some indications that it may be reaching a plateau in the developed world [52, 59]. Researchers have made progress uncovering the mechanisms and pathophysiology of asthma, leading to improved treatment and management. However, diagnosing asthma still remains a challenge for physicians [60–62], and misdiagnosis can lead to unnecessary treatment, greatly increased medical costs [63], or missed treatment for vulnerable populations such as the elderly [61, 62]. In this case, mitigating the hyperchemotactic activity of inflammatory cells could lead to improved clinical outcomes for asthmatic patients.
Cancer metastasis and asthma represent two diseases in which chemotaxis can play a central role in the pathophysiology of disease. Researchers have studied these pathways in an attempt not only to understand the biology of a disease, but also to develop targeted therapies. For instance, a therapy blocking the production of interleukin-5 (IL-5)—a cytokine that is implicated in mucus secretion, the recruitment of eosinophils to the lungs (leading to eosinophilia for asthmatic patients), and airway hyper-responsiveness (AHR)—might decrease eosinophil and mast cell recruitment to the lungs, resulting in symptom relief for the patient [64]. Anti-IL-5 clinical trials are currently underway [65, 66], demonstrating how therapeutic interventions can be developed once the underlying pathway is uncovered. In order to understand the role of cell chemotaxis in various diseases and facilitate the development of drug therapies, researchers require robust in vitro assays to controllably and systematically study cell chemotaxis. In the following sections, we will explore both traditional macroscale chemotaxis assays and more recently developed microfluidic systems. Furthermore, we will explore how properly designed microfluidic chemotaxis devices can potentially be used in a clinical setting for diagnostic and therapeutic purposes.
33.2.3 Traditional Methods for Studying Cell Chemotaxis
Several assays have been developed to study cellular chemotaxis (Fig. 33.2). The first widely adopted in vitro assay was developed by Stephen Boyden in 1962 [67], known as the "Boyden chamber" or "Transwell" assay. The Transwell assay is a two-chamber system, with each chamber filled with media and separated by a microporous membrane (Fig. 33.2a). The lower chamber contains a test substance of interest (e.g., chemoattractants), and the upper chamber contains cells. During the course of an experiment, the source compound diffuses across the porous membrane due to the concentration gradient that exists between the two chambers. Chemotactic cells sense the spatial distribution of chemoattractant and, depending on the cell type and chemoattractant, migrate across the membrane and toward the source. Multiple wells can be studied simultaneously and in parallel, allowing incorporation of proper controls (i.e., no concentration gradient) or to test varying doses in order to characterize the chemotactic response of the cell type–compound combination. Several early studies demonstrated the utility of having an in vitro chemotaxis assay by characterizing the chemotactic response of several cell types under various experimental conditions [67, 71–73]. Following the successes of the Boyden chamber, researchers sought to develop new designs that offered different approaches to measuring cell chemotaxis.
Fig. 33.2
Overview of macroscale chemotaxis assays that have been developed over the years. The timeline below each assay indicates the date the technology was invented below each assay (not to scale). The direction of the arrow indicates the direction of cell chemotaxis. The color shading indicates the formation of the chemical gradient of chemoattractant [67–70]. a Boyden/Transwell. b Zigmod. c Micropipette/Needle. d Dunn
Alternative in vitro chemotaxis assays that have been utilized in the biology community include the Zigmond chamber [68]; the Dunn chamber, which is a modification of the Zigmond chamber design [70]; the under-agarose assay [74]; and micropipette-based assays [69]. A comparison of some of these chemotaxis techniques is shown in Fig. 33.2. The Zigmond chamber consists of two etched wells—a chemoattractant (source) well and a well containing cells and media (sink)—that are separated by bridge that restricts convection of fluid from one well to the other (Fig. 33.2b). The cells are placed on an inverted coverslip spanning the two wells, allowing the investigator to observe cellular chemotaxis across the bridge in response to a chemical gradient. However, the gradients are short-lived (~1 h) and are somewhat unstable due to the evaporation that occurs in this design. The Dunn chamber design [70] sought to overcome these disadvantages and employs a spherical geometry, allowing the wells to be completely encapsulated by the coverslip to limit evaporation (Fig. 33.2d). The under-agarose assay is another early example of a technique that allowed the investigator to visually observe the migration of cells over time. In this assay, holes are punched in agarose gel, and a chemoattractant is placed in the holes to act as a source for the concentration gradient. Due to the concentration gradient between the source and the surrounding gel, the compound diffuses into the gel and cells sense the changing spatial distribution of the source compound over time and migrate toward the source under the agarose gel. This method is a simple and robust technique for creating one or more chemical gradients, while visualizing the response of cells in a physically confined environment. Foxmann et al. utilized the multisource capabilities of the under-agarose assay to characterize the neutrophil chemotactic function in competing gradients [75], and there are many other studies that utilize this technique [76, 77]. However, as useful as these in vitro chemotaxis assays have been over the years, researchers have discovered several limitations that limit their utility.
Technological limitations of these assays include the following: large sample volume requirements, unstable or unpredictable gradient profiles, and usability issues that make it difficult to run the desired experiment. For example, the most common macroscale chemotaxis assay in use today—the Transwell assay—develops unstable chemical gradient profiles [78]; filters cells by size and deformability due to the rigid microporous membrane the cells must cross [79]; and delivers ambiguous results as chemotaxis and chemokinesis are difficult to differentiate in this system [80]. The under-agarose assay allows for visualization of the cell migration path, but produces an unstable chemical gradient in all directions [78]. Additionally, under-agarose assays have low spatial resolution with respect to source and cell placement because of the way the experiment is conducted. The user is required to define these locations with a micropipet and crude hole-punching methods for the source of chemoattractant, both of which can be subject to significant variability. Therefore, while the method is relatively simple to perform and can be useful for visualizing cell migration paths, the under-agarose assay is not well-suited for reliable quantification of cell chemotaxis. Another popular and more modern visual chemotaxis assay is the Micropipette assay (also known as the "Needle assay"). This technique offers significant technical advances over these older techniques, such as high-resolution real-time imaging (Fig. 33.2c and [81]). However, the method is also best suited for qualitative analysis because its onerous labor requirements limit investigators to low-throughput sampling. Another significant limitation of most macroscale assays is that they require large sample volumes in order to isolate specific cell types of interest. For example, to perform a neutrophil chemotaxis experiment using these common macroscale techniques, the sample preparation includes a blood draw and cell purification protocols [82] requiring tens of milliliters of blood. This sample volume requirement limits the number of times a subject can be sampled to probe their neutrophil chemotactic function, thereby restricting the time resolution of the experiment. Additionally, the large blood volumes needed makes sampling infants or small animals (e.g., mice, rats, etc.) for neutrophil chemotaxis logistically complex, if not impossible.
Microfluidic technologies play an important role in providing solutions to many of these limitations. Indeed, over the last decade, microfluidic engineers have either solved or minimized the gradient stability and sample volume limitations of macroscale assays. What properties of microfluidic systems enable them to improve upon current chemotaxis assays? In the following sections, we will detail why microfluidic technologies are particularly useful for chemotaxis assays by exploring the physics of fluids at the microscale.
33.2.4 Microfluidic Methods for Studying Cell Chemotaxis
Microfluidic devices are ideally suited to perform chemotaxis assays and offer significant advantages over macroscale approaches. As previously described, chemotactic eukaryotic cells can sense spatial and temporal perturbations in their microenvironment, and respond by directionally migrating toward the increasing concentration of the stimulus. The cell cannot undergo chemotaxis, however if the concentration changes so suddenly that the receptors on the cell membrane are unable to discern a difference in soluble factors from one side of the cell to the other. Fortunately, the physics of fluids at the micro/nanoliter scale makes creating diffusion dominant mass transport a relatively trivial task. Fluids at this scale are often characterized by a series of dimensionless quantities [83]; the quantity that is most widely cited is the Reynolds number (Re, Eq. 33.1):
where v is the characteristic velocity of the fluid; L 0 is the characteristic length scale in which the fluid is traveling; and η is the kinematic viscosity of the fluid. For devices with dimensions on the micrometer scale that manipulate fluids with typical velocities (v ≪ m/s) and kinematic viscosity (e.g., aqueous solutions, oils, etc.), the Reynolds number, Re ≪ 1, is well under the transition from turbulent to laminar flow. Thus, the size scale of microfluidic technologies dictates that fluids generally behave according to the laminar flow regime. The implications of this phenomenon as applied to creating controllable gradients of soluble proteins are significant. Laminar fluids can be manipulated, combined, and separated in highly predictable and reproducible ways without the introduction of stochastic convective currents (turbulence). Specifically, two or more neighboring microfluidic streams can run adjacently to each other without turbulent mixing; instead, molecular diffusion dominates the mass transport between the laminar fluids.
Diffusion is a process governed by Brownian motion, where molecules move from a higher concentration to a lower concentration (i.e., thermodynamically driven). The kinetics of this process are determined by the activation energy required to move the diffusing molecule (e.g., proteins) through a particular medium (e.g., aqueous solution). In one dimension, diffusion can be modeled by (Eq. 33.2):
where d is the distance a molecule with diffusivity D travels in time t. There is another dimensionless quantity that describes the ratio of convection of a fluid compared to diffusion of a molecule within that fluid, known most commonly as the Peclet number (Eq. 33.3):
where v and L 0 are the characteristic velocity of the fluid and length scale of the system, respectively, and D is still the diffusivity of a molecule from one fluid into the medium of an adjacent fluid. This number helps to determine the relative importance of diffusion compared to the convection of the fluid. In practice, the Pe number can be used to determine the necessary length of a microchannel to allow a complete diffusion of a molecule from one fluid stream to another. For example, consider two fluids—Fluid A and Fluid B—flowing in adjacent laminar flow streams at 10 μm/s in a microchannel with width of 500 μm, and we wish to calculate the channel length required for a protein with diffusivity of 50 μm2/s to diffuse from Fluid B to the channel edge of Fluid A (Fig. 33.3). Due to the lack of convective mixing on this scale, the protein B will take 20 channel widths or 5 cm to diffuse across Fluid A (a distance a few times the diameter of a human hair). These numbers demonstrate that diffusion can be well controlled within microchannels, but without convection, transport of molecules from one area of a microchannel to another takes considerable time.
Fig. 33.3
Generic design of two fluids flowing through a microchannel in the laminar flow regime. Diffusion of molecules from fluid B occurs transverse to the fluid flow and down the concentration gradient toward fluid A
Microfluidic engineers have leveraged the unique properties of fluids at the microscale to make significant advancements in the capabilities of chemotaxis assays. Microfluidic devices are ideally suited tools for chemotaxis assays because they can simulate the diffusion-dominant phenomena present in the cellular microenvironment. A classic example of a microfluidic chemotaxis device that leverages microfluidic fluid phenomena is the "Christmas tree" design developed by George Whitesides' lab in 2000 [13]. In this system, multiple concentration gradients are created by mixing fluid streams with different source concentrations in serpentine microchannels (Fig. 33.4a), then recombining the fluids with different combinations of the source concentration once the serpentine channels meet downstream. The result is a user-defined concentration gradient of the original source molecules (Fig. 33.4b). This channel design exploits laminar flow phenomena to generate highly reproducible concentration gradients that are difficult or impossible to achieve using macroscale techniques. The laminar flow properties of the system allow for the components of each source fluid to be separated and then recombined in a predictable and controllable fashion. However, the lack of convection in the system means a serpentine channel design must be utilized to achieve complete mixing due to the high Peclet number. This channel design has been used for developing chemical gradients for multiple cell types, including neutrophils [13], neural stem cells [84], Dictyostelium discoideum [85], and metastatic breast cancer cells [86].
Fig. 33.4
Schematic and data of the microfluidic "Christmas tree" gradient generator. a Design of the serpentine gradient generator that shows the splitting and recombination of multiple fluid streams to create a chemical gradient. b Gradient profiles of fluorescent dye that show the versatility of the technique [88]
The "Christmas tree" design is one example of leveraging microfluidic phenomena to develop highly predictable chemical gradients; however, many other gradient devices have been developed over the years using microfluidics. For example, Wong et al. describe a microfluidic system that utilizes hydrogel barriers to compartmentalize the source, sink, and cell channels [87].
Here, the authors make use of properties of the hydrogel that enable its use in cell culture—namely the high viscosity of the hydrogel that prevents convective mass transport and the permeability of hydrogels, allowing protein diffusion from one compartment to another (e.g., from the source, to the cells, to the sink). In another class of static, no-flow microfluidic gradient generators, engineers have taken advantage of the greatly increased resistance to fluids as microchannel height is decreased:
where Q is the flow rate of the fluid, ∆P is the pressure drop across the microchannel, and R is the fluidic resistance of the microchannel. Equation 33.4 shows that the convection rate of the fluid is inversely proportional to the resistance in of the microchannel. For a microchannel with rectangular cross section and high aspect ratio (width of channel far greater/less than the height of the microchannel), the resistance in the microchannel is given by:
where h and w are the height and width of the channel, respectively; L is the length of the microchannel; and μ is the viscosity of the fluid [89]. From Eq. 33.5, we see that the resistance of a microchannel with a high aspect ratio is inversely proportional to the cube of its height. Therefore, a device can be engineered to be highly resistive to convection by decreasing the height of the microchannel (e.g., 7–15 μm) compared to the height of the cell, source, and sink channels. This technique of separating microchannels containing cells and reagents by low-height "diffusion channels" has been exploited to create no-flow gradient devices [90, 91] and for microfluidic multiculture systems [92]. This approach offers the benefits of highly controlled chemical gradients without requiring tubing or active pumping systems and conserves intercellular signaling that would normally be lost in a convection-based design. Other microfluidic designs have been reported that emphasize user-friendly operation to facilitate adoption by biologists, while generating robust chemical gradient profiles [93]. For example, our lab recently reported a two-component chemotaxis device that sorts neutrophils within minutes and can easily form a chemical gradient by placing a lid with chemoattractant onto a base where the neutrophils have been sorted [30]. This technique enabled a new set of applications that were difficult or impossible to conduct using macroscale chemotaxis assays, demonstrating how microfluidic systems can not only simplify, but also enhance the capabilities of biomedical researchers. The technological advancements in microfluidics combined with their demonstrated advantages over macroscale assays lead to another potential application, which is clinical diagnostic chemotaxis assays.
33.2.5 Microfluidic Chemotaxis for Translational Research
Assays that directly assess cellular chemotactic function have the potential to make valuable contributions to medicine. The concept of probing the function of a cell type to confirm disease state is beginning to show promising results. This approach seeks to elicit information from the cell type of interest in order to diagnose disease, or perhaps monitor drug therapies. However, the approach of using cell chemotaxis as a readout for clinical medicine is at an embryonic stage, with few studies demonstrating this approach to-date. The few existing published studies share several characteristics, including superior control of the biochemical gradient compared to macroscale chemotaxis assays; user-friendly operation helped by utilizing passive pumping techniques [94] for fluid handling and the use of visual readouts that track cell migration over time.
Neutrophils are a possible candidate cell type for eliciting disease information based on chemotaxis function because of their central role in the pathophysiology of several diseases, as well as their robust ability to undergo chemotaxis. Several studies have probed neutrophils for diagnostic information [90, 91, 95]. For example, Butler et al. described a neutrophil chemotaxis platform that analyzed neutrophils from burn patients to characterize their health status [91]. In this study, the authors found that the magnitude of the burn injury negatively correlated with the speed of neutrophil migration toward the chemoattractant and impaired neutrophil chemotaxis observed as early as 24 h after the patient was burned. This result validates trends observed in previous studies using traditional chemotaxis techniques, such as the Transwell and Zigmond assays [96], although those studies did not document changes in neutrophil function until 72 h after the patient was burned. Additionally, the authors found a correlation between total body surface area of the burn and neutrophil chemotaxis function of the patient. The improved detection of disease phenotypes for the burn patients compared to macroscale techniques is likely due to the improved sensitivity that microfluidic gradient generators can achieve. Keeping user-friendly design considerations in mind, the authors designed this microfluidic chemotaxis device to operate without any external fluid handling equipment (Fig. 33.5a). In contrast, most traditional macroscale chemotaxis assays would be difficult to operate in a clinical setting. This study demonstrates how microfluidic assessment of chemotactic function can provide rich, functional information that can potentially be used as a diagnostic, prognostic, and/or therapeutic biomarker.
Fig. 33.5
Recent user-friendly chemotaxis devices that have been utilized for clinical applications. a Neutrophil chemotaxis through cell-sized microchannels (3 μm × 6 μm) that allow for easy quantification, robust persistent neutrophil chemotaxis, and percent convection from the source to the sink [91]. b A chemotaxis device that also utilizes a low-height "diffusion channel" to prevent convection, along with a low-resistance "bypass" to direct any potential convection in the diffusion channel [90]. c A neutrophil chemotaxis device that operates by placing a lid containing chemoattractant onto a base containing the cells. Once the connection is made, the chemoattractant can diffuse into the microchannel where the cells are visually tracked over time. Experimental and modeling data of the gradient development are shown. Importantly, all three of these methods can be operated using only a micropipette, and do not require complex fluid handling equipment of expertise [30]
In our research lab, we have also explored the possibility of using neutrophil chemotaxis for diagnostic applications [90]. Berthier et al. describe a microfluidic chemotaxis device that can easily establish biochemical gradients in a high-throughput screen and automatically track neutrophil chemotactic function (Fig. 33.5b). This study analyzed the neutrophil chemotactic function from an infant who presented recurrent bacterial infections. The analysis showed a significantly retarded neutrophil chemotactic response for the patient compared to both healthy controls and the infant's parents (0.7 μm/s vs. 0.15–0.17 μm/s, respectively). The assay enabled analysis of changing neutrophil morphology and quantitative characterization of the neutrophil chemotaxis. Importantly, both pieces of information conveyed a signaling defect for the patient. Indeed, the patient was later diagnosed with a rare genetic mutation in a GTPase Rac2 (D57 N) that has been previously reported in the literature for an infant with immunodeficiency [97]. This sort of rare immunodeficiency is difficult to diagnose clinically, but this study demonstrates that a functional readout based on cell chemotaxis can potentially aid in making the diagnosis. In another clinical study, we are employing an adaptation of a previously published chemotaxis platform (Fig. 33.5c, [30]) to study whether neutrophil chemotaxis can be used as a biomarker to characterize or diagnose asthma. In this study, we have utilized a microfluidic kit design, where all the reagents required to run the assay are assembled in a complete, user-friendly assay. Additionally, the method employs a neutrophil sorting technique [93] that can be performed in several minutes using blood obtained from a lancet puncture. These features make it simple to rapidly perform the chemotaxis assay and make the system well suited for implementation in a clinical setting. Preliminary results from this study indicate that neutrophil chemotactic function may be impaired for asthmatic patients compared to non-asthmatic, allergic rhinitis patients. Although the results still need to be confirmed with additional experiments, the study illustrates how microfluidic solutions can provide significant advantages over macroscale assays for clinical applications. Furthermore, these reports demonstrate that chemotaxis can be a useful readout in the clinic to assist physicians with diagnosis or management of a variety of diseases.


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Presto News - 22nd April 2013
Pappano conducts Rossini's Petite messe solennelle
Rossini really knew how to live: having made a fortune penning operas at an astonishing pace during his twenties and thirties, he had pretty much retired from musical life by the time he was forty and spent the next twenty years or so indulging his passions for fine wines and cuisine (his creative legacy includes the indulgent dish 'Tournedos Rossini'!). But he didn't abandon music altogether: after leaving Italy for Paris in 1855, the salon culture of his new social circle led him to scribble over a hundred smaller-scale pieces which he ruefully dubbed his 'Péchés de vieillesse' or 'Sins of Old Age'. These 'sins' were intended only for domestic performance at 'soirées musicales' which he hosted with his second wife, the former model Olympe Pélissier (the glamorous guest-list often included Liszt and Meyerbeer). The most ambitious of Rossini's 'Péchés' was the Petite messe solennelle (thought to have been written for a recently-bereaved aristocratic family who frequented Rossini's soirées), but even this was still very much domestic in conception, being originally scored for just twelve singers, two pianos and harmonium.
Sir Antonio Pappano
I'm sure I won't be the first person to observe that the ensuing work is neither 'petite' nor especially 'solennelle' – Rossini clearly had his tongue firmly in his cheek in describing it as such, because most of the work brims over with sunny vitality, and even the most sombre sections are never small-scale or understated (especially in the orchestrated version, which the composer prepared himself in his last years, although it wasn't performed until after his death).
Though very popular with choral societies, Rossini's 'Little Mass' hasn't had all that many outings on disc – I first got to know the piece through an old Decca recording with Mirella Freni and Pavarotti – so I was very excited to receive this new set from Sir Antonio Pappano and his classy Santa Cecilia orchestra and chorus. Their blazingly theatrical recording of Rossini's Stabat Mater was one of my favourite discs of 2010, and was followed by an equally thrilling account of William Tell, so these musicians have serious pedigree when it comes to Rossini. Pappano's reading is bursting with brio and never overdoes the occasional moments of darkness in the score, but at times there are even distinct echoes of Verdi's Requiem – another sacred work from an opera-composer that's so theatrical in temperament you can practically smell the greasepaint.
One of the hallmarks of Pappano's previous recordings with these forces has been his happy knack of assembling solo quartets of apparently very disparate voices which somehow come together quite magically, and this musical alchemy is in evidence again here: he really has the balancing of light and dark timbres down to a fine art. I've been hearing great things about the young Latvian soprano Marina Rebeka over the past year or so: she's had several operatic triumphs recently when stepping in at short notice after cancellations by starrier singers, and on this showing her big, shiny voice will surely propel her into the firmament in the near future. Her bright, glossy tone is heard to great effect in the 'O salutaris hostia', and contrasts beautifully with Sara Mingardo's wine-dark, dusky contralto in the 'Qui tollis'. For me, though, the stand-out performance comes from tenor Francesco Meli, audaciously old-school with his liberal portamenti in the unapologetically jolly 'Domine Deus' and clearly having great fun in the process. This is a gorgeous, golden voice that seems to hark back to the gramophone era – shades of the young(ish) Pavarotti on that old Decca recording I mentioned earlier.
This vivacious account of an 'old man's folly' is just the disc to welcome in spring, then: listening to the irrepressible, bouncy 'Cum Sancto Spiritu', it's been nigh-on impossible to concur with T.S. Eliot that April is the cruellest month!
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Rossini: Petite messe solennelle
Marina Rebeka (soprano), Sara Mingardo (contralto), Francesco Meli (tenor), Alex Esposito (bass), Orchestra e Coro dell'Accademia Nazionale di Santa Cecilia, Sir Antonio Pappano
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Katherine Cooper - email@example.com
New Releases
22nd April 2013
This is just the pick of the recent releases. The New Releases and Future Releases pages are always available for browsing all the new and forthcoming releases.
Stravinsky: Le sacre du printemps
Berliner Philharmoniker, Sir Simon Rattle
Sir Simon Rattle and the Berliner Philharmoniker return with a recording of the ground-breaking ballet Le Sacre du printemps, whose premiere a century ago marked a turning point in 20th-century music. The programme on this release also includes new recordings of Stravinsky's Symphonies of Wind Instruments and Apollon Musagète.
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The Art of Magdalena Kožená
Magdalena Kožená (mezzo-soprano)
The Art of Magdalena Kožená is an anthology of her finest performances, documenting both the range of her voice and the breadth of repertoire to which she can bring authority, from the early baroque of Monteverdi and Strozzi to sacred arias by Bach, opera arias by Handel, Vivaldi, Mozart, Gounod, Bizet and Auber, as well as orchestral songs by Mahler.
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A Bach Notebook for Trumpet
Jonathan Freeman-Attwood (trumpet) & Daniel-Ben Pienaar (piano)
Jonathan Freeman-Attwood's 'A Bach Notebook for Trumpet' is a celebration of the music of the famous Johann Sebastian and ten other lesser-known Bachs, re-imagined for the combination of trumpet and piano. Bach specialist Daniel-Ben Pienaar has arranged music from a great uncle, close cousins, distant cousins, and five sons, all related to the great J.S. Bach in a wholly unique recital programme.
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Simon Trpčeski: Schubert, Bach, Liszt
Simon Trpčeski (piano)
Trpčeski's programme for this Wigmore Hall Live CD draws strongly on his deep immersion in national traditions of music and dance throughout his childhood. Schubert's tuneful 16 German Dances pave the way to what is arguably the composer's most virtuosic piece, his 'Wanderer' Fantasy. By way of a transcription of Bach's Prelude and Fugue in A minor BWV543, the second half of the programme focuses on Liszt.
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La Capella Reial de Catalunya: 25 Years
La Capella Reial de Catalunya, Jordi Savall
To celebrate the 25th anniversary of the Capella Reial de Catalunya, the choir he founded in 1988, Jordi Savall has gathered four examples of their work, rich in Catalan heritage, luxuriously presented in a box and all published originally on the Astrée label.
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Glass: Symphony No. 3 & The Hours
Manitoba Chamber Orchestra, Anne Manson
In this new recording made at Canada's famous Glenn Gould Studio at the CBC in Toronto, conductor Anne Manson collaborates with pianist Michael Riesman and her own Manitoba Chamber Orchestra in a tour de force performance of Glass's Oscar-nominated music from The Hours and a virtuosic performance of Glass's Third Symphony for Strings.
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Handel/Vivaldi: Dixit Dominus & In furore iustissimae irae
Lucy Crowe (soprano), La Nuova Musica, David Bates
David Bates directs La Nuova Musica in a pair of contrasting settings of Psalm 109. Handel's masterful and ambitious HWV282 was penned in 1707 during a youthful visit to Italy. Vivaldi's vivid and economical RV807 was long mistakenly attributed to Baldassare Galuppi. Rounding out the programme is Vivaldi's dazzling motet for solo voice, In furore iustissimae irae, featuring soprano Lucy Crowe.
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Jennifer Pike plays Brahms & Schumann
Jennifer Pike (violin) & Tom Poster (piano)
In 2002, at the age of twelve, Jennifer Pike notably became the youngest-ever winner of the BBC Young Musician of the Year competition. She has given performances throughout the UK and abroad and is widely regarded as one of the finest violinists in Britain. Tom Poster is internationally recognised as a pianist of outstanding artistry and versatility, equally in demand as a soloist and as a chamber musician across an unusually extensive repertoire.
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BBC Radio 3 CD Review
Saturday 20th April 2013
Building a Library - Debussy: Pelléas et Mélisande
First Choice
Wolfgang Holzmair (Pelléas), Anne Sofie von Otter (Mélisande), Laurent Naouri (Golaud), Orchestra National de France, Bernard Haitink
Disc of the Week
Berlioz: Grande Messe des Morts, Op. 5 (Requiem)
Barry Banks (tenor), London Symphony Orchestra, London Symphony Chorus & London Philharmonic Choir, Sir Colin Davis
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