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Q: Compare times on separate dates in Ruby/Rails? I have 3 different TimeWithZone objects:
from = Mon, 20 Apr 2020 00:00:00.000000000 UTC +00:00
to = Mon, 20 Apr 2020 23:59:59.000000000 UTC +00:00

event = Sat, 01 Jan 2000 07:00:00.000000000 UTC +00:00

I don't care about the dates. All I need is to be able to see if the time of event falls within the time bounds of from and to.
In the example above, the event does fall within the time bounds.
I've tried converting the from and to to just their time values using strftime and then converting them back to times with #to_time, but this causes the hours and minutes to change.
Any ideas?

A: You can format every value and than compare them
from_time, to_time, event_time = [from, to, event].map { |t| t.strftime("%H%M%S%N") }

event_time.between?(from_time, to_time)

Another option - compare just seconds (as Float)
from_sec, to_sec, event_sec = [from, to, event].map(&:seconds_since_midnight)

event_sec.between?(from_sec, to_sec)

redpajama_set_name: RedPajamaStackExchange


Purse Woven Pouch with Zipper hand woven manta aguayo material.
This is a colorful and delightful little purse. It is handcrafted from traditional Andean textile.
Bright color assortment and lined interior.
Easy to carry and open or close.


Place your bets on this crazy bug racing game. These formula 1 bugs don't just overtake by going around their opponents, they go over and under too and at super speed (if your device can handle it!).Place your stake on your chosen bug and see how much you can win, with good odds on pole position bugs down to back markers on the grid. It's a game for one player or up to eight people can play if you want, as winnings are shown for all bugs after each race.


Giving to the Lord, His Church and His purposes unleashes a joy that sets us free and brings breakthrough for others. We want to be free from greed and materialism so we can freely serve God (Matthew 6). In other words, we want to live simply, work diligently and give generously.
Use the button below for a one-time donation or to set up a recurring contribution to Antioch's missionaries serving around the world.


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Home News Cristiano Ronaldo opens his own hair transplant clinic
Cristiano Ronaldo opens his own hair transplant clinic
Tuesday, 19 March 2019, 11:42 am
Cristiano Ronaldo and his girlfriend Georgina Rodriguez have returned to Madrid in order to open their own hair transplant centre.
Looking pretty pleased with himself at the opening, the footballer beamed as he greeted fans at the clinic. The pair were dressed to the nines as they both rocked a rather clinical look in crisp white shirts.
Just recently the 34-year-old spoke about his reasons to start the new business, and joked about getting a hair transplant of his own.
He told ABC: 'Madrid is the city which made a mark on my life forever from a professional and emotional point of view.
'I developed an important part of my career there and it's also where I built my family. I like to support entrepreneurial projects with solid features to spread the best of what we have in Portugal throughout the world.'
Asked whether or not he'd have the procedure himself, the star said: 'When I think it's necessary [to have a transplant], of course I'll do it. 'One's image is an essential tool for being successful. For me it's fundamental.'
The clinic, which has been named Isparya, will offer both hair and beard transplants as well as eyebrow transplants.
But it seems Ronaldo will have a lot on his hands after opening the new business, as he also shares a baby daughter with Georgina.
Chatting about their little one, the 25-year-old revealed that having a baby together only made the pair stronger. She told Hello! Magazine: 'It has definitely brought us closer. We're happier together than ever.'
SOURCEmetro
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redpajama_set_name: RedPajamaCommonCrawl


Of falling on my face doing handstand walks.
Of falling on my face doing a ring muscle up.
Of hurting myself to the point I can't work out.
Of not pushing hard enough.
Of burn out (which happens quite frequently).
I'm glad I'm not the only one who goes between being afraid of doing too much and hurting myself and being afraid that I'm not doing enough!
It seem as though fear get a lot of attention in your post. I believe in healthy fear that causes us to contemplate our limits and helps us stay within them, By walking through unrealistic fear we can grow and become strong. I have learned to ignore the fear that try's to paralyze me by making me fearful of everything.
This is exactly my sons attitude … he has been pushing himself so much .. getting ready for Basic Training for Army National Guard . Sadly he is fit but still pushes himself so hard.
There is always choice. I am in no position to comment upon fitness and exercise but I can speak up in the name of common sense. Listen to what your body tells you – that fear is one of your own defence mechanisms in action; please do not ignore it!
Yes, you need to just keep going while paying attention to your body and its needs, so you do not overdo!


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1940 Dr. William 2022
Dr. William L. Bishop
March 9, 1940 — February 14, 2022
Dr. William L. Bishop, 81, went home to be with the Lord on February 14, 2022.
Born on March 9, 1940 he was the son of the late Rev. and Mrs. Vernon Bishop and the husband of "Jan" for 59 years! In addition to his parents, he was predeceased by his son, Brian William Bishop. "Dr. Bill" was a graduate of Palmer College of Chiropractic in Davenport, Iowa and practiced for 35 years in Cowpens. He served in the National Guard for 5 years in Gaffney and was a proud member of the U.S. Air Force for 4 ½ years,
Survivors also include three daughters, Tonya Stott and Eric of Campobello, Dana Jones of Arden, NC., Brandi Bishop of Spartanburg and several wonderful grandchildren, Tyler Stott, Tiffany Stott Page and Austin, Abby Jones, Noah Jones, Bridges Bishop, Maggie Bishop, and Lucie Bishop. Also surviving is his brother, Jim Bishop and Faye of Forest City, NC., and a sister Estelline Coyle and Earl of Gaffney.
The family will receive friends from 12:00 p.m. until 12:50 p.m. Friday, February 18, 2022 at Harris-Nadeau Mortuary. A celebration of life will follow at 1:00 p.m. in the Chapel of Harris-Nadeau Mortuary with Rev. Scott Scrimpsher and Rev. Kenny McDowell officiating.
The family is at the home.
In lieu of flowers, memorials may be made to Buck Creek Baptist Church Building Fund, 506 Buck Creek Road, Chesnee, SC 29323.
Online condolences may be expressed by visiting www.harrisnadeaumortuary.com
To order memorial trees or send flowers to the family in memory of Dr. William L. Bishop, please visit our flower store.
12:00 - 12:45pm (Eastern time)
Harris - Nadeau Mortuary
5880 Chesnee Hwy, Chesnee, SC 29323
Starts at 1:00pm (Eastern time)


NUMA, Film Production Equipment & Facilities, listed under "Film Production Equipment & Facilities" category, is located at 20 Teamsters Cl Port Douglas QLD, 4877, Australia and can be reached by 0740985200 phone number. NUMA has currently 0 reviews.
Browse all Film Production Equipment & Facilities in Port Douglas QLD.


/ 60 Seconds of Smart Home / Has Amazon Echo become a Dorm Essential? The Smart Dorm.
Guest post written by Level Up Your Home Super Intern, Monica Cipriano, incoming Sophomore at University of Maryland.
Is the Echo Dot Really Worth It?
The Amazon Echo Dot has generated a lot of buzz over the past year, claiming to act as your personal assistant. But does it live up to the hype? Recently I was given the opportunity to take a Dot home and test it out, to see if it really is a useful tool or just a glorified version of Siri.
As a college student, my main priority is to get my life together. College is an exciting place to try new things and figure out what you want to do, but it's also chaotic, busy, and overwhelming. Therefore, when I was exploring Alexa, I was looking for ways she can improve my life at school so I'm not constantly a jumbled mess. Here are my top 5 favorite things I discovered.
Traditional day planners only work if you remember to write everything down and remember to check them. With Alexa, you can set reminders, customize alarms, and schedule your week all without having to pick up a pen. This year my class times vary drastically, so having Alexa there to automatically adjust my alarm based on the day of the week will be so much better than manually adding and deleting alarms on my phone every night.
With Alexa, I can order anything I want without thinking twice. If I want pizza, she gets dominos. If I want coffee, Starbucks is a command away. An Uber? She does that too. By asking Alexa to order for me, I can save time when I am with my friends, and nobody needs to scramble to find a laptop. It's surprisingly life changing.
Alexa connects to Spotify! As someone who finally stopped mooching off their friends account and has gotten one of her own, I can say I use Spotify every day. Alexa can access my playlists, find new ones, and play the top songs of the week. Outside of Spotify, she gives you recommendations based on genre, plays the top hits, and even sings herself (which is very amusing).
Alexa can look up almost anything you ask her, making her one of the most helpful homework tools I know. Ask her math questions, science facts, you name it. You'd be surprised how much more you get done when you ask her questions rather than stopping what you're doing to manually type them into a search engine.
By far my favorite aspect of Alexa is that there is always more she can do. Alexa comes with the ability to enable different skills, allowing her to get smarter. You can enable games like Jeopardy, skills like ordering food, and funny anecdotes like telling jokes. It's impossible to grow bored of her.
The consensus? It's totally worth it.
Although it was skeptical at first, the Echo Dot is very useful. It exists to simplify your life, and it does its job well. The more you interact with Alexa, the more helpful she becomes, and she does quite literally become your personal assistant. Personally, I am very excited to bring her to school with me so she can help me get my life together.


Rest in peace to a hip-hop legend, The Notorious B.I.G.
Classic Rotation: The Notorious B.I.G.'s "Life After Death"
The Notorious B.I.G. predicted his own death and conquered the world on "Life After Death."
From Biggie's "I Got A Story To Tell" to Ice Cube's "Today Was a Good Day".
6 Artists You Forgot Were on "Life After Death"
The "Life After Death" features were crazy.
Which tale from the crypt is your favorite?


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Matt Carpenter, Cardinals agree to extension
The St. Louis Cardinals and infielder Matt Carpenter have agreed to a six-year, $52 million extension, according to media reports on Saturday. Ken Rosenthal originally reported on Thursday that Carpenter and the Cardinals were close to a six-year extension.
Carpenter's contract includes a $1.5 million signing bonus and an $18.5 million club option with a $2 million buyout. He is represented by Bryan Cahill of SSG Baseball. His contract is comparable to Andrew McCutchen's extension of $51.5 million two years ago.
"For a baseball junkie like myself, I can't think of a better place to play than the city of St. Louis," Carpenter said. "Busch Stadium, I still pinch myself every time I make that drive to the field. Playing in front of these fans, the city of St. Louis, Cardinal baseball as a whole, it's just so much fun. It's such a privilege to put on this jersey. It's something I don't take lightly at all."
The 28-year-old Carpenter has been an anchor in the Cardinals lineup since he came into the Major Leagues in 2012. In 2013, he led the National League in runs scored, hits, and doubles, while appearing in his first career All Star Game. He hit a triple-slash line of .318/.392/.481 with 11 home runs and 78 RBI (143 OPS+). Carpenter posted an excellent oWAR of 6.7.
Over his career, Carpenter has hit a triple-slash line of .306/.381/.470 with 17 home runs and 124 RBI. He's posted a 8.1 oWAR and a dWAR of -0.7 over his 278 games. Carpenter, who has made $984,000 over his career, will get a huge contract, something that his excellence has been deserving. Carpenter will be a force in the Cardinals lineup for years to come.
The St. Louis Cardinals have made sure they will be keeping a good piece in their lineup through at least 2019. They've done a fantastic job by locking Carpenter up, and they should be rewarded with his great hitting.


The average human's salt intake should only be ideally one teaspoon a day, but we all know that with the types of food the average person eats, the majority are ridiculously over their recommended sodium intake.
When choosing what to eat, go for fruit and vegetables as much as possible as they are low in sodium content. This is the same for canned and frozen fruits/vegetables also. Try and eat fresh meats over packaged meats. Packaged meats contain more sodium, and of course… Fresh is always best! If you are worried about your salt intake, check food labels when doing your weekly shop. Manufacturers are legally required to let you know exactly how much of what, you are consuming. Anything labelled in red means it contains more than the daily recommended amount.


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Home News Reports point to failed Indigenous policies
Reports point to failed Indigenous policies
By Shannon Jenkins
A new report from the Centre for Aboriginal Economic Policy Research (CAEPR) says Australia is failing to produce effective Indigenous policy.
It comes on the same day as an audit report which revealed the Indigenous Advancement Strategy's evaluation framework is only in its "early stages" despite the strategy being introduced five years ago.
"In relation to Indigenous affairs policy, the nation has currently reached a dead end," Neil Westbury and Michael Dillon, authors of the CAEPR paper, said.
They argue that "continuing systemic social, economic and political exclusion" of Indigenous Australians creates a discontinuity between policy objectives and policy outcomes. Such exclusion from "mainstream institutional frameworks" and the pursuit of prejudiced policies harm national unity and the ability to effectively address issues.
"Over the last 30 years, no Australian Government has developed, resourced and effectively implemented a comprehensive overarching long-term policy reform strategy in Indigenous affairs," argue Westbury and Dillon, who combine a wealth of experience of Indigenous Australia, both inside and outside the public sector.
"Current policy settings are not fit for purpose, and governments appear bereft of ideas or proposals capable of addressing the deep-seated challenges that exist."
The termination of ATSIC and the implementation of the 2007 Northern Territory intervention confirmed the death of Australia's acceptance of self-determination, according to the report.
The CAEPR paper suggests the only way to overcome such deep-seated policy failure is through:
Structural or systemic reform.
Ongoing engagement with key actors to minimise the risk of loss of influence and to maximise the scope of influence.
Comprehensive strategy by Indigenous interests aimed at identifying key institutional barriers and institutional locations of Indigenous exclusion.
Implementing sustained long-term policy and political campaigns to have such institutions adjusted, reformed or abolished.
Developing and sustaining alliances with key mainstream interests to increase the likelihood of success in whichever political strategies they choose.
Governments arguing that the ongoing failures and inability to recognise the legitimacy of Indigenous voices will continue to damage the national interest.
READ MORE: PM&C sets high standards for Indigenous affairs evaluation
The scathing assessment of Indigenous policy was released on Tuesday, the same day as a new report from the auditor-general, which seems to confirm their argument.
The audit looked at how effectively the IAS evaluation framework ensures high quality, ethical and inclusive evaluation, to improve outcomes for Indigenous Australians.
The Department of the Prime Minister and Cabinet's implementation and management of the framework is only partially effective and faced "substantial delays", ANAO found.
"As the department is still developing procedures to support the application of the IAS evaluation framework, it is too early to assess whether evaluations are being conducted in accordance with the framework," ANAO said.
The audit made several recommendations, suggesting PM&C use reliable methodology and develop a comprehensive set of procedures to implement the IAS evaluation framework.
It also suggested the department develop structured criteria for assessing significance, contribution and risk and conduct a strategic analysis of gaps in evaluation coverage.
READ MORE: Marcia Langton: Government accelerating Indigenous people into 'permanent poverty'
Shadow Ministers Linda Burney and Warren Snowdon criticised the strategy following the audit's release.
"Indigenous Australians deserve better," they said.
"The Government is establishing a new agency within the existing department to manage Indigenous programs – but it will take much more than a name change to fix the deep-seated problems with the IAS."
The Greens and the National Aboriginal Community Controlled Health Organisation also voiced their concerns over the findings.
Shannon Jenkins is a journalist at The Mandarin based in Melbourne.
Departments: Australian National Audit Office Centre for Aboriginal Economic Policy Research Department of the Prime Minister and Cabinet
Tags: ANAO Audit CAEPR Indigenous Advancement Strategy indigenous affairs


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As one of the highlights, Zimbabwe offers a visit of the impressive Victoria Falls. Wonderful and diverse safaris could also be experienced in this beautiful country. Hwange National Park for example is known for its dense population of elephants, whereby Matusadona National Park is known for its beautiful scenery at Lake Kariba. Mana Pools National Park is an untouched paradise for unique safari experiences on land as well as on the majestic waters of the Zambezi River.
Why Zimbabwe
The widest waterfall in the world is annually visited by thousands of tourists. Victoria Falls is situated between the border towns Livingstone in Zambia and Victoria Falls in Zimbabwe. The falls have a width of almost two kilometres and are also locally called 'The smoke that thunders'. In the wet months, there are about 550 million litres of water that fall down about 120 m per minute. Enjoy the spectacular views during a helicopter ride over the Victoria Falls. You get a completely new perspective from the bird's eye view and realize how huge and impressive this natural wonder is. We also recommend a guided walk along the Victoria Falls, which takes about two hours.
Hwange National Park is situated just 120 km south of the Victoria Falls. Around 100 types of wild animals and 400 species of birds call the park their home. You could also wonderfully observe large herds of animals in Hwange National Park. Due to its proximity to the Victoria Falls, the park is also easily reached by road transfer.
Matusadona National Park and Lake Kariba
Lake Kariba is a large artificial lake and lies in the mainly untouched territory of the Matusadona National Park in the northeast of the country. Here you experience the charm and magic of this lake, which is surrounded by majestic hills and savannah. The park boasts a dense population of wild animals and is particularly known for its rhino population. Due to its location you could experience classic safaris by vehicle or by foot and boat safaris are especially good to view the wildlife on the shore of the lake.
VFA EL
Wildlife Kudu EL
Wildlife Rhino Head EL
Elephant Mum and Baby WL
At The Elephant Camp near the Victoria Falls you have the possibility to get in close contact with these impressive animals. A special attraction is an Elephant Back Safari, where you take part in a tour through the wilderness on the back of an elephant.
thousands of flying ants swarm out at Lake Kariba shortly after the beginning of the rainy season? Just the strongest of them survive to build new colonies. This spectacle is particularly interesting for fishermen, as the flying ants are the preferred bait for several sorts of fish. This means that successful fishing at this period is almost guaranteed within a very short time.
In Zimbabwe I recommend a stay in Matusadona National Park close to Lake Kariba. You easily reach this destination by air from Harare, Victoria Falls or Hwange. At the lake you have the possibility to do boat trips apart from classic safaris. During such you could observe elephants on the shore or listen to a concert of the hippos from a safe distance. Enjoy this perfect moment during sunset with a glass of wine, rounding off your varied and unforgettable journey.
Zimbabwe is the perfect country for a fly-in safari. You could ideally combine this with a visit to Victoria Falls. It's also worth considering the combination of one or two safari destinations in Zimbabwe with a shorter stay in Botswana. With such a combination you would be able to stay within a certain budget, if you like to have comfortable lodges and absolutely combine the Okavango Delta with a nice safari in other areas.
Republic of Zimbabwe
1 US$ = 100 cents as well as 1 South African Rand (ZAR) = 100 cent
390.757 km² (World rank 59), thereof 22,5% forest 7% farmland, 12% pasture land
West-East: 800 km, North-South: 750 km
11,529 million
Harare (1.184.169), Bulawayo (620.936), Chitungwiza (274.035), Mutare (131.808), Gweru (124.735)
Even though Zimbabwe is situated in the tropics, its climate is milder due its altitude. Winter is dry and often chilly. Rain is mostly limited to the hot summer months. The most rainfall occurs in the eastern mountain region.
Matetsi Victoria Falls
www.matetsivictoriafalls.com
Matetsi Victoria Falls is located in the private Matetsi Game Reserve, which is located halfway between Victoria Falls and Kasane. The transfer from Victoria Falls Airport to Matetsi Victoria Falls takes about an hour. The private Matetsi Game Reserve covers approximately 50.000 ha and has a 15 km private section of the Zambezi River on which wonderfully idyllic boat trips can be undertaken.
The lodge consists of two identical camps, each with 9 suites. One of these suites in each camp is a family suite. From the centre of the lodge, there are nine suites in both directions along the river, so all suites offer wonderful views of the Zambezi River. A private bathroom with both indoor and outdoor shower and bathtub, a private veranda with plunge pool and a seating area are provided with each suite. These are individually and comfortably furnished and built by using local materials and inspired by the tradition of fishing- and wickerwork in the area. Each wing of the camp has its own guest and dining areas, an interactive kitchen, bar and fire pit. The two family suites each consist of two sleeping areas with their own bathroom and are connected by a covered or closed bridge. Private verandas and plunge pool are also part of the family suites.
The common area of the lodge also features a fitness centre, massage area, swimming pool and a little curio shop.
Matetsi Game Reserve is home to large herds of buffalo and elephant, antelope, lion, leopard, wild dog, hyena, hippo and about 350 different species of birds. During your stay at Matetsi Victoria Falls, guided game drives, guided bush walks, sunset cruises on the Zambezi River, canoeing and fishing are included. Guided tours to Victoria Falls can also be arranged as an optional activity.
Singita Pamushana Lodge
Malilangwe Wildlife Reserve, Zimbabwe
Pamushana Lodge is located within the Malilangwe Wildlife Reserve in south eastern Zimbabwe. Built under shady trees the Pamushana Lodge blends in perfectly with the surrounding landscape. The Malilangwe Wildlife Reserve is set on about 60.700 ha of wilderness bordering the Gonarezhou National Park. The lodge is set amongst towering trees overlooking a picturesque lake in the remote reaches of the pristine reserve.
Accommodation is offered in 6 luxury suites (4x 1 bedroom suites, 1x 2 bedroom suites and 1x 3 bedroom suite) which offer fascinating views of the Malilangwe Dam. All master bedrooms feature en-suite bathroom and shower as well as an outdoor shower, air conditioning, overhead fans, private plunge pool, double-sided fireplace, mini-bar and fridge, room safe, private lounge, direct dial telephones and game viewing deck with Swarovski spotting scope. The architecture of the suites is inspired by the bold and vibrant culture of the local Shangaan people who have lived in the region for thousands of years. The public area offers an indoor dining room, open dining room, teak-deck dining areas, bar, library, open-air lounge, wine cellar, two heated swimming pools and a Jacuzzi.
The separately located Pamushana Villa offers beautiful views onto the Lake Malilangwe and features an additional 5 en-suite bedrooms, a dining room, a living room, a small kitchen and a private infinity pool. The villa is ideal for family and friends travelling together and can only be booked on exclusive use. It naturally comes with a private guide, chef and hosts.
Enjoy extensive day and night game drives through the private Malilangwe Wildlife Reserve, which is also home to rare species such as roan and sable antelope as well as to the black rhino. The area is famous for its cathedral Mopane forests, majestic 'upside-down' Baobab trees and rock sites, which date back more than 2.000 years. The lodge also offers guided walks, community visits, rock art site visits, fishing, archery and sundowner cruises on the lake. Optional activities are black rhino tracking and guided walks with Bushmen. There is a spa and a yoga room, two tennis courts as well as a gym at the lodge. A 9-hole golf course is just 45 minutes drive away.


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Chain Gang
By Izzy Grinspan Jul 31, 2008, 3:52pm EDT
Share All sharing options for: Chain Gang
The chain store with the most locations in New York City? Not Starbucks, though for now it does dominate Manhattan with 186 stores. Instead, the franchise that owns all five boroughs is Dunkin' Donuts, according to the Center for an Urban Future's "New York By the Numbers" survey. Also of note: There are 105 Sleepy's in NYC. Apparently all those donuts are making us tired. [Center for an Urban Future]


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Home News State By State 2019 Elections: Osun APC Candidate Accuses Reps Member, Police of Complicity in...
2019 Elections: Osun APC Candidate Accuses Reps Member, Police of Complicity in Attack
By Yetunde Oladejo, Osogbo
A House of Representatives candidate of the All Progressives Congress, (APC) in Obokun/Oriade Federal constituency of Osun State, Pharmacist Siji Olamiju, has accused the opposition party in the state, the Peoples Democratic Party (PDP) of attacking his campaign team on Tuesday.
Specifically, Olamiju pointed accusing finger on his co-contestant, Hon. Oluwole Oke, who is currently representing the federal constituency at the House of Representatives, of being the brain behind the attack.
Briefing newsmen in Osogbo on Wednesday, Olamiju also accused the police particularly, the Divisional Police Officer (DPO) in Ibokun, who was simply identified as Mr. Taofeek of complicity in the war against him and members of his party.
He explained that his members and campaign vehicles were attacked at Ase-Ijesa in which two persons were seriously injured by dangerous cultists allegedly recruited by Oke.
According to him, " We are about to approach the palace of the Alase of Ase-Ijesha when we got the news that some of our members were under attack, we quickly went back to ascertain the degree of injury and attend to the victims.
"When we got to the scene, we realised that two of our members had been stabbed by broken bottles and seriously injured, and because we are law abiding people, we directed the victims to go and make formal complain at a police station located in Ibokun.
"We were later called to be informed that instead of the DPO to allow those victims to make entry, they were surprisingly locked up for no reason.
"We went back there, we met police shooting and our campaign bus has been vandalised, having been riddled with bullet. and we demanded for explanation which he declined, even after I contacted the Commissioner of Police and he gave directive for the people to be released, the DPO flagrantly flouted the order", Olamiju said.
He said the police also attempted to kill the Chairman of Oriade Local Government, Hon. Obeisun, when he shot at him.
He said it is not safe to conduct election in Obokun/Oriade, stating that the police have already compromised.
He further alleged that Oke, alongside his cohorts publicly celebrated delivery of ammunition for the purpose of election eleven days ago at Esa-Oke, his home town.
Olamiju therefore called for high powered police investigative team to unravel the connivance between the police and a member of the lower chamber to ascertain the level of complicity and avert further break down of law and order.
When the police was contacted, the Commissioner of Police, Mr Fimihan Adeoye, said investigation has commenced on the matter.
He vowed that whoever is found culpable will face the wrath of law, saying that police will not delay taking them to court.
All efforts to get reaction of Hon Wole Oke proved futile as he did not answer calls nor reply text messages sent to his mobile phone at the time of filing this report.
All Progressive Congress
Obokun/Oriade Federal Constituency
Osun State
Pharmacist Siji Olamiju
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KWHA Constitutes C'ttee to Probe illegal Govt Properties Acquisition


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Gene expression map of the Arabidopsis shoot apical meristem stem cell niche
Ram Kishor Yadav, Thomas Girke, Sumana Pasala, Mingtang Xie, and G. Venugopala Reddy
PNAS March 24, 2009 106 (12) 4941-4946; https://doi.org/10.1073/pnas.0900843106
Ram Kishor Yadav
Thomas Girke
Sumana Pasala
Mingtang Xie
G. Venugopala Reddy
For correspondence: venug@ucr.edu
Communicated by Elliot M. Meyerowitz, California Institute of Technology, Pasadena, CA, January 27, 2009 (received for review December 9, 2008)
Despite the central importance of stem cells in plant growth and development, the molecular signatures associated with them have not been revealed. Shoot apical meristems (SAMs) harbor a small set of stem cells located at the tip of each plant and they are surrounded by several million differentiating cells. This imposes a major limitation in isolating pure populations of stem cells for genomic analyses. We have developed a system to isolate pure populations of distinct cell types of the SAMs, including stem cells. We have used this system to profile gene expression from 4 different cell samples of SAMs. The cell sample-specific gene expression profiling has resulted in a high-resolution gene expression map to reveal gene expression networks specific to individual spatial domains of SAMs. We demonstrate that the cell sample-specific expression profiling is sensitive in identifying rare transcripts expressed in a few specific subsets of cells of SAMs. Our extensive RNA in situ analysis reveals that the expression map can be used as a predictive tool in analyzing the spatial expression patterns of genes and it has led to the identification of unique gene expression patterns within the SAMs. Furthermore, our work reveals an enrichment of DNA repair and chromatin modification pathways in stem cells suggesting that maintenance of genome stability and flexible chromatin may be crucial for stem cell function. The gene expression map should guide future reverse genetics experiments, high-resolution analyses of cell–cell communication networks and epigenetic modifications.
CLAVATA3
fluorescence-activated cell sorting
WUSCHEL
Spatiotemporal regulation of transcriptional programs mediated by cell–cell communication networks is crucial in stem cell maintenance (1, 2). Understanding the complexity of transcriptional programs requires a high resolution analysis of gene expression, preferably at a cell type-specific resolution (3). Shoot apical meristems (SAMs) of higher plants represent a dynamic and interconnected network of distinct cell types. In Arabidopsis thaliana, the SAM consists of ≈35 stem cells, located within the central zone (CZ) (4). Stem cells are surrounded by several million differentiating cells that are part of the adjacent peripheral zone (PZ) and developing organs. The cells of the Rib-meristem (RM) that are located just beneath the CZ provide positional cues necessary for stem cell maintenance (Fig. 1 D and E). Earlier studies have revealed molecular mechanisms involved in stem cell maintenance (5). However, gene networks that underlie the stem cell maintenance process are far from understood. The development of high resolution gene expression map of distinct cell types of SAMs could enable network analysis of pathways involved in stem cell maintenance.
Expression profiling studies of specific cell types have been performed on Arabidopsis root system (3, 6), however, studies on the SAMs have been restricted to experiments of the entire tissue (7). This is because the domains of specific cell types of the SAMs are extremely small. For example, the stem cell domain marked by the expression of the only available marker, pCLAVATA3(CLV3)::mGFP5-ER [CLAVATA3 promoter attached to the endoplasmic reticulum localized-GFP], has revealed that there are only ≈35 stem cells within a SAM (Fig. S1 A and C). This imposes a limitation in isolating sufficient amounts of specific cell types for expression profiling (4). In this study, we have used apetala1-1;cauliflower1-1 (ap1-1;cal1-1) double mutant background to isolate fluorescently-labeled protoplasts from 4 distinct cell samples of SAMs. A single ap1-1;cal1-1 double mutant plant can produce up to several hundred SAMs owing to the conversion of floral meristem identity into inflorescence meristem identity, thereby providing primary enrichment of SAM tissue (Fig. 1 G–I) (8). The expression profiling of transcripts from 4 different cell populations and informatics analysis has revealed gene expression networks specific to different spatial domains of the stem cell niche. We demonstrate that the expression profiling method is sensitive in identifying rare transcripts expressed in a few specific subsets of SAMs. Our extensive RNA in situ analysis reveals that the expression map can be used as a predictive tool in analyzing the expression patterns of unknown genes (Fig. S2). Our work also reveals an enrichment of DNA repair and chromatin modification pathways in stem cells implying that maintenance of genome stability and flexible chromatin may be crucial for stem cell function.
Gene Expression and Cell–Cell Communication Machinery in ap1-1;cal1-1 System.
We tested whether ap1-1;cal1-1 SAMs are suitable for cell type specific expression profiling by analyzing the organization of the CZ and the RM. The expression patterns of CLV3, and WUSCHEL (WUS), markers for the cells of the CZ and the RM respectively, were visualized by whole mount RNA in situ analysis. This allowed us to demonstrate that the expression patterns of CLV3 and WUS are similar to the WT expression patterns observed in previous studies (Fig. S1 A and B) (9, 10). Furthermore, we tested whether the ap1-1;cal1-1 SAMs are comparable to WT SAMs with respect to the CLV-WUS-mediated intercellular communication process. Mutations in clv3 gene result in expansion of the CZ. CLAVATA3 (CLV3), a small extracellular protein synthesized in the CZ activates CLAVATA1 (CLV1)-CLAVATA2 (CLV2) receptor kinase complex in RM cells (5). The active CLV1-CLV2 receptor kinase complex functions by down regulating the expression of WUSCHEL (WUS), a homeodomain protein expressed in RM cells. An earlier live-imaging study has revealed that the transient silencing of the CLV3 gene results in an increased CLV3 promoter activity in the native domain within 24 h of CLV3 silencing and followed by the radial expansion of the CZ in the next 48 h. We tested whether a similar CZ reorganization pattern could be observed in the SAMs of ap1-1;cal1-1 plants. The live-imaging of SAMs of ap1-1;cal1-1 mutants, upon CLV3 silencing, revealed a similar temporal sequence of CZ expansion that was observed in WT SAMs (4). This suggested that the intercellular communication-mediated by CLV-WUS network is operational in ap1-1;cal1-1 SAMs (Fig. S1 C and D). Therefore, we adapted a protoplasting method, followed by FACS analysis to isolate fluorescently-labeled cell populations of distinct cell types from ap1-1;cal1-1 SAMs (11) (SI Text).
Description of Cell Type-Specific Fluorescent Markers.
We introduced fluorescent reporters into the ap1-1;cal1-1 background to differentially label 3 distinct cell types of the SAM stem cell niche. The pCLV3::mGFP5-ER was used to label cell types of the CZ (4). The CLV3 expression is restricted to the CZ and the expression extends into the 4th layer starting from the tip (Fig. 1 A and D). pWUS::mGFP5-ER (WUSCHEL promoter driving the expression of endoplasmic reticulum localized-GFP) was used to label cells of the RM and it is expressed only in a few centrally-located cells of the L3 layers (Fig. 1 B and E and Fig. S3D). The pFILAMENTOUSFLOWER (FIL)::dsRED-N7 (FIL promoter driving the expression of nuclear-localized dsRED) was used to label specific subsets of cells of the flower organ primordia located within the PZ (Fig. 1 C and F) (12). For simplicity, the 3 cell samples will be referred to as CLV3p, FILp and WUSp. The 3 fluorescent reporters do not express in all cell types of SAMs. Therefore, while collecting GFP positive protoplasts from CLV3p cells, we also collected GFP negative (CLV3n) protoplasts (Fig. S4). The CLV3n cell populations are expected to represent a mixture of SAM cell types that lacks the CLV3p cells.
Expression patterns of 3 marker lines of shoot apical meristems. (A) A 3D view of WT SAM labeled with FM4–64 (red) and pCLV3::mGFP5-ER (green), a marker for the central zone (CZ). (B) An optical section depicting pWUS::mGFP5-ER expression (green) and FM4–64 (red) in the WT SAM. (C) A 3D-view of WT SAM expressing pFIL::Ds-Red-N7 (red), marking the organ primordia. (D) A side-view of (A) exhibiting the pCLV3::mGFP5-ER expression extending up to the 4th cell layer from the tip. (E) A side-view of B, depicting the expression pattern of pWUS::mGFP5-ER (green) in the L3 layer (arrows) and cell layers located beneath. (F) A side-view of C, exhibiting the expression pattern of pFIL::Ds-Red-N7 (Red) (arrows). (G) A 3D-view of ap1-1;cal1-1 SAM labeled with FM4–64 (red) and pCLV3::mGFP5-ER (green) marking the CZ (arrows). (H) A horizontal sections of ap1-1;cal1-1 SAM expressing pWUS::mGFP5-ER (green) is labeled with FM4–64 (Red) depicting the expression of WUS in the RM (arrows). (I) A 3D view of the ap1-1;cal1-1 SAM exhibiting the expression of pFIL::Ds-Red (Red) marking the primordia in the shoot apex (arrows). (J) A comparison of MAS5-normalized microarray intensity values of 3 genes (CLV3, WUS, and FIL) across 3 cell types. (K) A comparison of relative levels of CLV3, WUS, and FIL transcripts, measured by qRT-PCR, in RNA samples derived from CLV3p, WUSp, and FILp cells respectively.
RNA samples from 4 different protoplast populations (CLV3p, FILp, WUSp and CLV3n) were hybridized to the Affymetrix GeneChip ATH1 in biological replicates. Correlation coefficients close to 1.0 among the replicates indicated a high reproducibility of the experiments (Table S1). To estimate the extent of gene expression changes in response to the protoplasting method, we compared the gene expression patterns of protoplasted-ap1-1;cal1-1 SAMs with nonprotoplasted-SAMs. Three hundred genes were found to respond to the protoplasting method. In the subsequent analysis these genes were not considered as cell-specific gene candidates (Table S2).
Cell Sample-Specific Expression Profiling Reveals Higher Sensitivity.
The cell type-specific transcriptome analysis of Arabidopsis root cell types has revealed higher sensitivity than experiments with whole root samples (7). To assess a similar sensitivity increase for our datasets, we compared the detectable gene sets obtained from cell sample-specific expression profiling dataset (cell-sorted) with that of whole (cell-unsorted) ap1-1;cal1-1 SAMs (Table S3). The comparison of cell-sorted data with that of the whole ap1-1;ca1-1 SAMs revealed 952 genes that could only be identified in the cell type-specific transcriptome (Fig. 2D). This cell type-specific set includes CLV3 (Fig. S5 and Table S4), MEI2 C-Terminal RRM-like1 (MCT1) (Fig. 3E and Fig. S2), B3-TF (Fig. 3H and Fig. S2) and JAGGED LATERAL ORGANS (Fig. S5 and Table S4), which are expressed in small subsets of cells within the SAMs. We also compared the detectable gene sets from our cell sample-specific data with that of the pooled-microarray data of entire AtGenExpress developmental series of WT plants (Fig. 2D and SI Text) (7). This comparison yielded 1031 genes that were unique to cell sample-specific data and it includes PUMILIO10 (Fig. 3A), CLV3, MCT1 (Fig. 3E), MCT2 (Fig. 3B) and BLADE ON PETIOLE2 (Fig. S5 and Table S4), all of them are expressed in very few cells of the SAMs. The comparison of detectable genes in our cell sample-specific dataset with that of the Arabidopsis roots revealed 3,915 genes specific to the root cell types, whereas 1,739 genes were specific to SAM cell types (Fig. 2D and SI Text) (3, 6). Taken together, the cell type-specific transcriptome, as expected, revealed higher sensitivity in identifying rare transcripts and genes that are unique to either the shoot or root cell-types.
Differential gene expression patterns of SAM cell types. (A) A sketch representing 5 regions of SAMs, the central zone narrow (CZ) (red), the CZ broad (green), the rib meristem (RM) (blue), the peripheral zone (PZ) (cyan), and the L1 layer (black). This sketch is a broad representation of different spatial patterns of characterized differentially-expressed genes. (B) Hierarchical clustering of 47 characterized differentially-expressed genes within the SAM, using the microarray data derived from 3 cell types (CLV3p, FILp, and WUSp). The color scheme used for representing clusters on the tree is same as the one used for representing different regions in A. (C) A 3-way comparison of all differentially-expressed genes enriched genes in CLV3p, WUSp, and FILp cell samples. The DEGs were classified into 6 sectors (SI Text). (D) A 4-way comparison of total number of genes detected (MAS5 present calls) in cell type-specific transcriptome (ap1-1;cal1-1 cell sorted), the whole ap1-1;cal1-1 apex (unsorted), the pooled microarray data of development series of WT plants from AtGenExpress study (AtGenExpress) and the cell type-specific data from Arabidopsis root tissue (roots). (E) Relative percentage of selected Gene Ontology (GO) groups identified in different DEG sectors. Note the number above each column represents a sum of total number of genes detected for a particular GO term in various sectors.
Validation of differentially-expressed genes reveals gene expression domains within the WT SAMs. Shown are longitudinal sections of WT SAMs revealing RNA in situ hybridization signals of 23 genes that belong to different sectors and intensity interval clusters. The gene identification numbers, the DEG sectors and the intensity interval clusters are marked. Refer to Fig. S2 for additional views of expression patterns. For example, the expression pattern in O could also be seen in deeper layers of SAMs.
Differential Expression Analysis and Validation of Microarray Data.
To identify differences in gene expression programs among the individual cell types, we identified differentially-expressed genes (DEGs) (SI Text). Systematic comparisons among all 3 cell samples identified a union set of 2515 DEGs [P < 0.01 (Table S5 and SI Text)]. Next we generated 3 lines of evidence to validate the cell sample specific transcriptome data. First, we measured the relative abundance of transcripts of 3 marker genes (CLV3, WUS, and FIL), in isolated protoplasts by quantitative RT-PCR (qRT-PCR). The results showed an increased expression of the transcripts for CLV3, WUS, and FIL in the respective cell samples, which correlates well with the microarray expression data (Fig. 1 J and K). The detection of WUS transcripts in CLV3p cells suggested that the expression of 2 genes overlap with each other. We generated a double transgenic plant carrying pCLV3::mGFP5-ER and pWUS::dsRED-N7 (WUSCHEL promoter attached to nuclear-localized dsRED) to test whether the expression domains of CLV3 and WUS overlap with each other. A high resolution microscopic analysis revealed that the 2 genes are expressed in overlapping sets of cells located in L3 layers of SAMs (Fig. S3 A–I). Although we detected high levels of WUS transcripts in CLV3p cells, however, a similar enrichment of WUS transcripts in CLV3p cells was not observed (Fig. 1 J and K). This could be due to the dilution of CLV3-expressing cells because of a higher ratio of WUSp:CLV3p cells in regions where the 2 domains overlap. Enrichment of WUS transcripts in CLV3p cells, although the overlap is restricted to very few cells, could be because of higher levels of WUS expression in the most centrally-located cells of the WUS expression domain that overlap with the CLV3 expression domain. Our whole-mount WUS RNA in situ expression patterns reveal that WUS is expressed in a graded fashion with higher levels of expression detected in the centrally-located cells (Fig. S3 I and J). Second, we tested the transcript abundance of 7 genes by qRT-PCR that appeared to be differentially-expressed among CLV3p and CLV3n cell samples and found that the relative transcript abundance in the respective cell types was in agreement with the microarray data (Fig. S4). Third, we analyzed the relative expression differences of genes for which RNA in situ expression patterns and promoter reporter expression data are available from earlier studies (Fig. 2B, Fig. S5, and Table S4). The relative enrichment of characterized differentially-expressed genes across cell samples was in agreement with our DEG analysis results (Table S5 and Fig. S5). Hierarchical cluster analysis grouped these genes largely according to their expression patterns observed on tissue sections, such as the PZ, the L1 layer, the CZ (includes narrow and broad) and the overlapping region between the CZ and the RM (Fig. 2 A and B).
Comparison of Differential Gene Expression Among 3 Cell Samples Reveals Unique and Overlapping Sectors of Gene Activity.
We organized the set of 2515 DEGs into 3 subcategories by determining genes that exhibited a significant up-regulation in 1 of the 3 cell samples (CLV3p, WUSp, and FILp) relative to at least 1 of the 2 other cell samples. The 3-way Venn diagram representation of these 3 DEG lists revealed 7 possible overlap/uniqueness sectors (Fig. 2C, Table S5, and SI Text). To mine this dataset, we evaluated the distribution of characterized marker genes in the individual Venn diagram sectors. The sector S5 (CLV3 expression domain, which excludes WUS and FIL expression domains) is expected to represent the central part of superficial cell layers (the L1 or the L2 or both the L1 and the L2) of the SAM and it includes CLV3, AINTEGUMENTA-LIKE (AIL5, AIL6, and AIL7) and TERMINAL EAR LIKE1 (Table S5 and Table S4). The sector S4 (shared between CLV3 and WUS expression domains, which excludes FIL domain) is expected to represent central part of the SAM including the superficial cell layers of the Rib-meristem and it includes SPATULA, REPRODUCTIVE MERISTEM1, HANBA TARANU/MONOPOLE, CLAVATA1, WUS, SHOOTMERISTEMLESS, TERMINAL FLOWER1, CYTOKININ OXIDASE5, PIN FORMED1 (Table S5 and Table S4). The S6 (WUS expression domain, which excludes CLV3 and FIL domains) is expected to represent deeper cell layers of differentiating region of the Rib-meristem. The S2 sector (shared between CLV3 and FIL expression domains, which excludes WUS domains) is expected to represent superficial cell layers (the L1 or the L2 or both the L1 and the L2) of the entire SAM surface including differentiating cells of the organ primordia and it includes ARABIDOPSIS CRINKLY4, ARABIDOPSIS THALIANA MERISTEM LAYER1, PROTODERMAL FACTOR 1 and 2, and BODYGUARD (Table S5 and Table S4). The S7 sector (exclusive FIL expression domain) is expected to represent cells of the PZ including differentiating organ primordia and it includes FIL, ASYMMETRIC LEAVES1 and 2, CUP SHAPED COTYLEDON3 and many such genes (Table S5 and Table S4). The S3 (shared between FIL and WUS expression domains, which excludes CLV3 domain) is expected to represent a relatively broad SAM region encompassing the differentiating cells of the organ primordia and the deeper cell layers of the Rib-meristem, and BREVIPEDICELLUS is associated with this sector (Table S5 and Table S4). Because a large part of the CLV3n cell sample is expected to be a part of FILp and WUSp cell samples, it was not included in the DEG analysis. However, a side-by-side comparison of expression values of genes in CLV3n cell sample with other cell samples has enabled us in assessing the radial dimensions of expression domains of genes that are part of the central region of SAMs (Fig. S2 and Fig. S5). Taken together, our analysis demonstrates that the dataset is suitable for analyzing the gene expression programs enriched in distinct spatial domains of SAMs.
Subclassification of DEGs Based on Expression Intensity.
The organization of SAMs has been described with the help of a limited set of molecular markers. New molecular markers are required to gain a better understanding of the spatial organization of SAMs. Therefore, we sought to identify markers for cell types of SAMs by mining the datasets obtained from DEG analyses. DEG analyses, however, provide information across a continuous range of expression levels. To predict genes that are only expressed in discrete subdomains of the SAM, we subclassified the identified DEGs by their expression levels in the individual cell samples (Fig. 2C and 4A). For this, their normalized intensity values from the 3 cell samples, CLV3p, FILp and WUSp, were subgrouped into the 4 expression states absent (A), low (L), medium (M), and high (H) (Fig. 4A and SI Text). The 4 expression intervals for the 3 samples provide 81 possible intensity interval clusters (IICs), of which only 49 were observed in the dataset of the 2515 DEGs (Table S5 and Table S6). Approximately 42% of the DEGs could be classified into 3 IICs with the most uniform expression patterns, AAA, LLL and MMM (Fig. 4B and Table S6). The remaining 58% of DEGs received different intensity intervals, among cell types, and they could be organized into 2 subclasses (Fig. 4B and Table S6). The first one contained 12 IICs accounting for 47% of DEGs and the rest 11% of DEGs belonged to the remaining 34 IICs suggesting that their spatial expression is relatively restricted to specific subsets of cells (Fig. 4B and Table S6). Consistent with this hypothesis, a majority (40/47) of characterized differentially-expressed genes could be found in the latter 2 subclasses, suggesting that this approach can be applied to identify genes that are expressed in specific subsets of cells (Table S5 and Table S6).
Subclassification of DEGs by expression levels. (A) The gene expression levels of the identified DEGs were subdivided into 4 intensity levels, using the following intervals of the MAS5 normalized intensity data: A = absent (<220); L = low (220–1,000), M = medium (1,001–5,000) and H = high (>5,000). (B) A pie chart showing the relative distribution of selected groups of intensity interval clusters in the dataset. The sample order is defined as CLV3p, FILp, and WUSp, respectively (for example, AAA means absent in all 3, CLV3p, FILp, and WUSp samples). (C) The relative distribution of 12 intensity interval clusters that account for the 47% of differentially expressed gene are mapped onto individual DEG sectors derived from Fig. 2C. (The relative distribution of 49 intensity interval clusters that account for all DEGs can be found in Table S6). Above column is the total number of genes that are part of various intensity interval clusters (IICs) of a given sector. The relative distribution of different IICs, of a given sector, is expressed as percentage.
Validation of DEGs by RNA in Situ Analysis Reveals Markers for Stem Cell Niche and Unique Expression Domains.
Next, we mapped the 49 IICs onto the 6 overlap/uniqueness sectors of the DEG analysis (Fig. 4C and Table S6). This allowed us to predict genes with discrete spatial expression patterns and confirm their expression patterns by RNA in situ analysis (Fig. 3). For example, we hypothesized that the genes belonging to the LAA (Low in CLV3p, Absent in FILp and Absent in WUSp) cluster in the S4 and S5 sectors should exhibit an overlapping expression with the CLV3 domain (Fig. 4C and Table S5). Indeed, the in situ experiments confirmed such an overlapping expression pattern within the CLV3 domain (Fig. 3 A–F and Fig. S2). The subtle variations to the CLV3 pattern could be observed, with gene expression restricted specifically to the L1 layer of the CZ (Fig. 3 C and F) or to the L2 layer of the CZ (Fig. 3D). The genes that are shared between CLV3 and WUS domains could be identified in LAL, MAM, MAL, LAH or MLM cluster of S4 sectors (Fig. 3). These genes exhibited, the expression that overlaps with both the CLV3 and the WUS domains (Fig. 3 G–Q and Fig. S2). The genes that were part of ALM, LLM and AAL clusters exhibited expression within the Rib-meristem (Fig. 3 R–T). Genes expressed in specific subsets of cells of the PZ could be found in the S7 sector (Fig. 3U and Fig. S2). A few genes that are part of the CLV3n cell sample, as expected, were excluded from 3 cell types and their expression was detected in the vasculature cells (Fig. 3 V and W and Fig. S2). Taken together, differential expression analysis and subsequent classification of DEGs based on expression levels can provide a better idea about their spatial expression patterns. However, the approach may not resolve finer differences in spatial expression patterns of genes that belong to the same IIC (Fig. 3 K and Q). This could be because of the inherent differences in gene expression levels and the rigid cut-off scale used for defining the intensity intervals. Nevertheless, our extensive in situ experiments demonstrate that the 49 IICs are a valuable data source for predicting spatial expression patterns. By using the markers that are expressed in distinct cell layers of the CZ (Fig. 3 C and D), we may be able to improve the spatial resolution and predictive capability of our expression map in the future.
Assessment of Overall Accuracy of the Technique and the DEG Analysis.
To assess the accuracy of the technique, we considered a total of 70 documented differentially-expressed genes, including 23 expression patterns described in this study, whose spatial expression patterns could be judged with a fair degree of confidence. A comparison of DEG-predicted sectors of these genes with their observed spatial expression patterns on tissue sections revealed that 4 genes were incorrectly assigned to different DEG sectors and the other 4 genes were not identified as differentially-expressed, resulting in a combined error of 11% (Table S4). In contrast to our expectations, BARELY ANY MERISTEM1, CUP SHAPED COTYLEDON2, REVOLUTA and ULTRAPETALA1 were not identified as DEGs, although a relative enrichment of these genes in expected cell samples could be detected (Table S5, Table S4, and Fig. S5). A notable exception is CUP SHAPED COTYLEDON1, which was associated with the S5 sector (CLV3p) instead of the S7 (FILp) (Table S4 and Table S5).
Overrepresentation of DNA Repair and Epigenetic Pathways in Stem Cells.
It has been speculated, based on the organization of cell types, that the plant stem cell niches may share common properties with that of animal stem cell niches (13). However, molecular evidence to support this view is largely lacking because of the nonavailability of stem cell specific gene expression profile. To assess the molecular composition and pathways enriched stem cells, we performed enrichment analyses of Gene Ontology (GO) terms in our dataset (Fig. 2E and Table S7). First we tested the DEG sectors for overrepresented-GO terms that are characteristic for certain cell types of SAMs. The sector S2, which contained genes, expressed in the L1 layer was enriched with genes that are involved in wax biosynthesis (P < 1.9 × 10−2) and lipid metabolism (P < 1.2 × 10−2), which is consistent with one of the expected function of the L1 layer cells in secretion of epi-cuticular wax (14). As expected, the differentiating cells of the rib-meristem (S6) and differentiating cells of developing organs (S7) were over-represented with genes involved in various metabolic processes including that of carbohydrate metabolism, lignin deposition and photosynthetic genes (Fig. 2E and Table S7).
The sector S5, which is enriched in meristematic genes is over-represented with genes involved in DNA metabolism (P < 1.17 × 10−17), and DNA replication and repair (P < 8.41 × 10−6, 6.72 × 10−5) (Fig. 2E and Table S7). This includes MUTLI PROTEIN HOMOLOG 1 (ATMSH1), MUTS HOMOLOG 2 (MSH2), MUTS-LIKE PROTEIN 4 (MSH4), MUTS HOMOLOG 6-2 (MSH6-2), RADIATION SENSITIVE (ATRAD17), RADIATION SENSITIVE (ATRAD51), ARABIDOPSIS THALIANA BREAST CANCER ASSOCIATED RING1 (ATBARD1), ARABIDOPSIS THALIANA BREAST CANNCER SUSCEPTIBILITIY1 (ATBRCA1), and ARABIDOPSIS THALIANA TELOMERASE REVERSE TRANSCRIPTASE (ATTERT), involved in telomere maintenance. The enrichment of both the enzymes of the mismatch repair and nonhomologous end-joining (NHEJ) DNA repair pathways suggest that the SAM stem cells exhibit typical characteristics of cells experiencing stress (15). It is conceivable that error-free DNA replication is critical to prevent incorrect cellular amplification of mutations and amplification of stem cell daughters that carry double strand breaks, both can be deleterious to plant development.
Genes involved in chromosome organization and biogenesis (P < 4.84 × 10−7) were also over-represented in stem cells suggesting that the stem cell chromatin is maintained in a flexible state (Fig. 2E and Table S7). They include genes involved in HISTONE and DNA modifications such as centromeric HISTONE H3, HISTONE ACETYL TRANSFERASE (HAM1), HISTONE DEACETYLASE 18 (HD18), HISTONE LYSINE AND METHYL TRANSFERASES (SUVH4, SUVH9, and SUVR2), ARABIDOPSIS TRITHORAX-LIKE 1 (ATX1) and SIRTUINS (SIR1 and SIR2), NAD-dependent deacetylases. Recent studies on murine embryonic stem cells (ES) have identified bivalent domains of both H3 lysine 27 methylation (a repressive chromatin mark) and H3 lysine 4 methylation (a chromatin mark that positively regulates transcription) within genes that encode developmentally important transcription factors. This suggested that the bivalent domains are responsible for keeping differentiation genes silent in ES cells, while keeping them primed for activation (16, 17). The enrichment of DNA and HISTONE modifying enzymes in stem cells of SAMs suggests that they might play a similar role in maintaining flexible chromatin to facilitate dynamic balance of gene expression. This can be tested, in future experiments, by profiling DNA and HISTONE modifications of individual cell types of SAMs with the method described here.
The genomic instability and changes in gene expression are regarded as hallmarks of the aging process in eukaryotes. A recent study, on mouse embryonic stem cells, has revealed that the mammalian deacetylase, Sir2 (SIRT1), in response to DNA damage, relocalizes to the sites of DNA breaks to mediate repair. The over-expression of SIRT1 has been shown to promote survival of mouse model of genomic instability and suppress age-related changes in transcription (18). The overrepresentation of SIRTUINS (SIR1 and SIR2), pathways that respond to DNA damage and chromatin-modifying proteins suggests that the stem cells are under selective pressure to correct mutations.
We have described a method to generate a cell sample-specific gene expression map of SAMs. Our work has identified many genes with cell sample-specific expression profiles within the SAM stem cell niche. The gene expression patterns identified in this study can be used to refine the spatial organization of cell types of the SAMs. Identification of molecular signatures associated with these cell types may provide clues to their function. The markers for cells of the stem cell niche, identified in this study, can be a valuable resource in following the dynamic regulation of stem cell maintenance by cell–cell communication. The dataset is a valuable resource for guiding future functional analyses of stem cell enriched genes including evolutionarily conserved PUMILIO class of translational repressors and genes homologous to LONELY GUY (LOG) of rice, which has been shown to function in meristem development by regulating cytokinin biosynthesis (Fig. 3 A and K) (19, 20). More significantly, it is now possible to analyze cell–cell communication networks at a single cell type resolution, similar to the studies carried out in the Arabidopsis root system (21, 22). Elucidation of molecular networks at higher spatiotemporal resolution may provide a molecular framework to model cell–cell communication processes within the SAMs (23).
Transgenic Lines.
The cell-type specific fluorescent reporter constructs used for expression profiling experiments have been described in the following studies, CLV3::mGFP5-ER (4) and FIL::dsRED-N7 (12). pWUS::mGFP5-ER reporter line was assembled in pZP222 vector by using a 4.5-kb fragment upstream of WUSCHEL ORF and a 1.5-kb fragment downstream of the stop codon. The transgenic lines were generated using floral dip method (24). The double transgenic line expressing CLV3::mGFP5-ER and WUS::dsRED-N7 has been described earlier (25). The transient silencing of CLV3 was achieved by using the constructs described in earlier study (4).
Protoplasting of SAM Cells.
Approximately 200 SAMs of 24-day-old ap1-1;cal1-1 plants were harvested within 15 min and placed in protoplasting solution for ≈1 h and 15 min. The concentration and composition of cell wall digesting enzymes were optimized to obtain maximum protoplast yield (11, 26). Harvested shoot apices were incubated in protoplasting solution [Solution B = Solution A + 1.5% cellulase (YAKULT; catalog no. 203039), 1% Pectolyase (YAKULT pharmaceutical Ltd. Tokyo Japan, Catalog no. 202047) and 1% Hemicellulase (Sigma USA, catalog no. H2125)], within a 70 μM cell strainer placed in a small Petri-plate. Subsequent steps involved in harvesting protoplasts for FACS-mediated separation were carried out according to an earlier study (6). The modified protoplasting method yielded ≈150,000 to 200,000 fluorescently labeled protoplasts. The purity of sorting events was independently confirmed through microscopic observation of sorted protoplasts.
We thank Barbara Walter (Institute of Inegrative Genome Biology, University of California, Riverside) for help with the FACS sorting and microarray hybridizations, Zhenhua Deng for help with qRT-PCR analysis, Nayana Hegde for help in generating marker lines, and Elliot Meyerowitz for providing FIL::dsRED-N7 DNA construct, Nicholas Provart and Zhenbiao Yang for comments on the manuscript. DNA sequencing/quantitative PCR/Microarray data were generated by the Genomics Core at the Institute for Integrative Genome Biology at the University of California, Riverside. This work was funded by National Science Foundation Grant IOS-0718046 (to G.V.R.).
1To whom correspondence should be addressed. E-mail: venug{at}ucr.edu
Author contributions: R.K.Y., S.P., M.X., and G.V.R. performed research; R.K.Y., T.G., and G.V.R. analyzed data; R.K.Y., T.G., and G.V.R. wrote the paper; G.V.R. designed research.
This article contains supporting information online at www.pnas.org/cgi/content/full/0900843106/DCSupplemental.
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You are going to email the following Gene expression map of the Arabidopsis shoot apical meristem stem cell niche
Ram Kishor Yadav, Thomas Girke, Sumana Pasala, Mingtang Xie, G. Venugopala Reddy
Proceedings of the National Academy of Sciences Mar 2009, 106 (12) 4941-4946; DOI: 10.1073/pnas.0900843106


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69-1903. Distribution for promotional purposes prohibited.
(1) Manufacturers, wholesalers, or retailers, or their representatives, of smokeless tobacco products shall not distribute for promotional purposes.
(2) Evidence of distribution of smokeless tobacco products to the general public shall be prima facie evidence of distribution for promotional purposes.
Source:Laws 1989, LB 48, § 3.


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Detroit Automakers Mold Their Future
The previous year's losses and gains in the automotive arena are already reported. On one side, the industry's victors are celebrating their success. On the other side of it, a significant few are lamenting. Conversely, every storm must come to an end and the respite must be used for the betterment of the industry. Now, Detroit automakers are in the process of recuperating from the ill effects of market shifts and evident crisis.
Earlier, Chrysler opened the billion-worth Jefferson North Assembly Plant that mirrored the travails and triumphs of Detroit – the Motor City. Lee Iacocca reluctantly opened the factory in 1992, despite forewarning from experts who said it was crazy to build an assembly plant in the inner city with an aging work force. However, Jefferson North persisted and the result was overwhelming.
Jefferson North blends the best of Chrysler and Mercedes. Said offer captures the fancy of auto aficionados. As a fact, the hot-selling Jeep Grand Cherokee produced by the plant exceeded the owner's expectations. Fortunately, it pumped out billions of dollars plus a breath of new life on the financial ropes just after experiencing a near-bankruptcy difficulty. In the previous years up to the present time, auto competition is downright intense. It was coupled with rising petrol prices and the boom of diverse types of vehicles. With the production falling, the plant seems a likely object for shutting. Chrysler prepares to announce that its restructuring will affect the plant. The restructure will likely pave way for the rebirth of Jefferson North as the automaker unfolds its own renaissance in the coming years.
The restructuring plan calls for the Auburn Hills automaker to jointly develop more vehicles with Mercedes Benz - its sibling within Germany-based DaimlerChrysler AG. This information was divulged by individuals familiar with the plan. In view thereof, the next-generation Grand Cherokee will be built off the vehicle architecture designed for the Mercedes M-Class SUV and go into production about 2010 at the said plant in Jefferson North. The upcoming Dodge Durango SUV will be built off the same architecture at Jefferson North. Other vehicles like the forthcoming Chrysler Aspen and other Jeep vehicles will also be manufactured in said factory.
If the automaker's plan comes to fruition, Jefferson North, once dubbed "the plant that never should have been built," could get a multimillion dollar investment and play a great role in yet another comeback in the boom of Chrysler. "It's a pretty significant deal for the plant itself," said Jeff Schuster, global forecasting analyst for J.D. Power and Associates in Troy. "(DaimlerChrysler) putting faith in the plant by bringing in Mercedes says a lot. It's giving the plant a vote of confidence."
"Iaccoca said he didn't think it was feasible building a plant in the city with a bunch of grandfathers building cars," said Aaron Taylor, the former president of United Auto Workers Local 7 who is now working for the UAW International as a service representative for Region 1. And he emphasizes, "We begged to differ."
"I had to convince the membership to drop about 144 classifications down to about 10," Taylor said. "I told them that our survival hinges on this agreement and that if we could not get this, the plant would not be built."
"We try to think positive," said Robert Ledgerwood, a nine-year veteran at Jefferson North. "Hopefully, Chrysler can continue to make some quality vehicles that people want to buy. That's the most important thing."
Ford, on the other hand, is also aimed at producing quality lineup to end its miseries in the industry. It is not only concentrating on how to improve the Volvo CV joint but the enhancements cover its entire brands and makes. Powerful lineup coupled with courage and positive vibes could pave way for the most-wanted change for the Detroit automakers.
About the Author: Glady Reign is a 32 year old is a consultant for an automotive firm based in Detroit, Mi. she is a native of the motor city and grew up around cars hence her expertise in the automotive field.


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